EP3887544A2

EP3887544A2 - Mirnas as biomarkers for training control and optimization

Info

Publication number: EP3887544A2
Application number: EP19832289.3A
Authority: EP
Inventors: Andreas Keller
Original assignee: Universitaet des Saarlandes
Current assignee: Universitaet des Saarlandes
Priority date: 2018-11-28
Filing date: 2019-11-28
Publication date: 2021-10-06
Also published as: WO2020109456A3; WO2020109456A2

Abstract

The present invention relates to a method of monitoring or determining the fitness level of a subject. The present invention further relates to a method of determining the training mode of a subject. The present invention also relates to a method of optimizing personal training of a subject. In addition, the present invention relates to a kit for carrying out the above-described methods.

Description

MIRNAS AS BIOMARKERS FOR TRAINING CONTROL AND OPTIMIZATION

BACKGROUND OF THE INVENTION

Fitness plays a very important role for a large part of the population. Physical activity helps to prevent life-style related diseases, such as cardiovascular diseases, metabolic diseases, and diabetes. Levels of physical activity that increase overall fitness may, therefore, be one of the most important modifiable life-style interventions indicated for preventing or controlling cardiovascular diseases, metabolic disease, and diabetes. Exercise training results in a large number of effects that benefit the subject in both health and disease. Endurance training, for example, increases VC max, improves lipid profile, improves endothelial function and increases abundance of capillaries, mitochondria and metabolic enzymes in different organs and systems. Strength training, for example, improves physical performance, improves movement control, improves walking speed, improves functional independence, and increases muscle strength and mass.

It has been found that micro RNAs (miRNAs) play a promising role as diagnostic and prognostic markers for human pathologies. However, miRNAs have also been correlated to different exercise modes and the overall physical performance capacity. In particular, the association of changes in miRNA level with physical activities has previously been reported.

Fitness training is primarily performed on the basis of universal exercise plans. Such a training is unindividualized and, thus, not always promising. There exists an unmet need for an individualized efficient training control and adaptation system. The common scheme„one-size- fits-all“ needs to be improved using a personalized approach. Although personal trainers offer the possibility of training individually, they are both expensive and working with them means being bound to a set timetable.

The present invention meets the above described need. The present inventors identified miRNAs which allow the determination and monitoring of the fitness level of a person in a cheap, simple and effective way. They also allow the determination of the training mode/type of training and the optimization of the personal training of a person. Thus, the present inventors developed a solution that is geared to the needs of people, it can be integrated into person’s daily working, studying or household schedules. It is specially tailored to and individually designed for a person. It ensures lasting wellbeing, and in particular, prevents cardiovascular or other common diseases.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to method of monitoring the fitness level of a subject comprising the steps of:

(i) determining the level of at least one miRNA in a blood sample of a subject at a first point in time,

(ii) determining the level of the at least one miRNA in at least one further blood sample of the subject at a later point in time, and

(iii) comparing said levels determined at the different time points,

wherein the at least one miRNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34.

In a second aspect, the present invention relates to a method of determining the fitness level of a subject comprising the steps of:

(i) determining the level of at least one miRNAs in a blood sample of a subject, and

(ii) comparing the level of the at least one miRNA to a reference level,

In a third aspect, the present invention relates to a method of determining the (predominant) training mode/type of training of a subject comprising the steps of:

(ii) comparing the level of the at least one miRNA to a reference level,

wherein the at least one miRNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 30, and SEQ ID NO: 32 to SEQ ID NO: 34.

In a fourth aspect, the present invention relates to a method of optimizing personal training of a subject comprising the steps of:

(ii) comparing the level of the at least one miRNA to a reference level, wherein the at least one miRNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 30, and SEQ ID NO: 32 to SEQ ID NO: 34.

In a fifth aspect, the present invention relates to the use of at least one polynucleotide for detecting at least one miRNA in a blood sample isolated from a subject for monitoring the fitness level of the subject,

In a sixth aspect, the present invention relates to the use of at least one polynucleotide for detecting at least one miRNA in a blood sample isolated from a subject for determining the fitness level of the subject,

In a seventh aspect, the present invention relates to the use of at least one polynucleotide for detecting at least one miRNA in a blood sample isolated from a subject for determining the training mode/type of training of the subject,

In an eighth aspect, the present invention relates to the use of at least one polynucleotide for detecting at least one miRNA in a blood sample isolated from a subject for optimizing personal training of the subject,

In a ninth aspect, the present invention relates to a kit for monitoring the fitness level or determining the fitness level of a subject comprising:

(i) means for determining the level of at least one miRNA in a blood sample of a subject, and

(ii) optionally at least one reference,

In a tenth aspect, the present invention relates to a kit for determining the training mode/type of training or optimizing personal training of a subject comprising: (i) means for determining the level of at least one miRNA in a blood sample of a subject, and

(ii) optionally at least one reference,

In an eleventh aspect, the present invention relates to the use of a kit for monitoring the fitness level, determining the fitness level, determining the training mode/type of training or optimizing personal training of a subject comprising:

(i) a microsampling device,

(ii) optionally an access code, and

(iii) optionally instructions on how to use the kit.

This summary of the invention does not necessarily describe all features of the present invention. Other embodiments will become apparent from a review of the ensuing detailed description.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in“A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number sequence submissions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

The term“comprise” or variations such as“comprises” or“comprising” according to the present invention means the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The term“consisting essentially of’ according to the present invention means the inclusion of a stated integer or group of integers, while excluding modifications or other integers which would materially affect or alter the stated integer. The term“consisting of’ or variations such as“consists of’ according to the present invention means the inclusion of a stated integer or group of integers and the exclusion of any other integer or group of integers.

The terms“a” and“an” and“the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

The term“miRNA” (the designation“microRNA” is also possible), as used herein, refers to a single- stranded RNA molecule of at least 10 nucleotides and of not more than 45 nucleotides covalently linked together. Preferably, the polynucleotides used in the present invention are molecules of 10 to 45 nucleotides or 15 to 35 nucleotides in length, more preferably of 16 to 28 nucleotides or 18 to 23 nucleotides in length, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,

41, 42, 43, 44, or 45 nucleotides in length, not including optionally labels and/or elongated sequences (e.g. biotin stretches).

The miRNAs regulate gene expression and are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (i.e. miRNAs are non-coding RNAs). The genes encoding miRNAs are longer than the processed mature miRNA molecules. The miRNA is initially transcribed as a longer precursor molecule (>1000 nucleotides long) called a primary miRNA transcript (pri-miRNA). Pri-miRNAs have hairpin structures that are processed by the Drosha enzyme (as part of the microprocessor complex). After Drosha processing, the pri-miRNAs are only 60-100 nucleotides long, and are called precursor miRNAs (pre-miRNAs). At this point, the pre-miRNA is exported to the cytoplasm, where it encounters the Dicer enzyme. Dicer cuts the miRNA in two, resulting in duplexed miRNA strands. Traditionally, only one of these miRNA arms was considered important in gene regulation: the arm that is destined to be loaded into the RNA-induced silencing complex (RISC), and occurs at a higher concentration in the cell. This is often called the“guide” strand and is designated as miR. The other arm is called the“minor miRNA” or“passenger miRNA”, and is often designated as miR*. It was thought that passenger miRNAs were completely degraded, but deep sequencing studies have found that some minor miRNAs persist and in fact have a functional role in gene regulation. Due to these developments, the naming convention has shifted. Instead of the miR/miR* name scheme, a miR-5p/miR-3p nomenclature has been adopted. By the new system, the 5’ arm of the miRNA is always designated miR-5p and the 3’ arm is miR-3p. The present nomenclature is as follows: The prefix“miR” is followed by a dash and a number, the latter often indicating order of naming. For example, hsa-miR-16 was named and likely discovered prior to hsa-miR-342. A capitalized“miR-” refers to the mature forms of the miRNA (e.g. hsa-miR-16-5p and hsa-miR-16-3p), while the uncapitalized“mir-” refers to the pre-miRNA and the pri-miRNA (e.g. hsa-mir-16), and“MIR” refers to the gene that encodes them. However, as this is a recent change, literature will often refer to the original miR/miR* names. After processing, the duplexed miRNA strands are loaded onto an Argonaute (AGO) protein to form a precursor to the RISC. The complex causes the duplex to unwind and the passenger RNA strand is discarded, leaving behind a mature RISC carrying the mature, single stranded miRNA. The miRNA remains part of the RISC as it silences the expression of its target genes. While this is the canonical pathway for miRNA biogenesis, a variety of others have been discovered. These include Drosha-independent pathways (such as the mirtron pathway, snoRNA-derived pathway, and shRNA-derived pathway) and Dicer-independent pathways (such as one that relies on AGO for cleavage, and another which is dependent on tRNaseZ). Further, the term“miRNA”, as used in this context, comprises not only the known miRNAs as e.g. annotated in the miRBase (see next definition) but also other small non-coding RNAs. These are not necessarily processed by the canonical miRNA processing pathway but other enzymes could be involved in maturing the molecules. Specifically, the set of miRNAs contains nucleic acid chains with the same or very similar properties as miRNAs that have been discovered by the inventors from over 2,000 blood data sets containing 100 billion small RNA reads. These can contain nucleic acid chains that are also similar to other non-coding RNA species such as piRNAs. The nucleic acid chains have been detected from the billions of reads by using the software miRMaster that has been recently developed by the inventors.

The term“miRBase”, as used herein, refers to a well-established repository of validated miRNAs. The miRBase (www.mirbase.org) is a searchable database of published miRNA sequences and annotation. Each entry in the miRBase Sequence database represents a predicted hairpin portion of a miRNA transcript (termed mir in the database), with information on the location and sequence of the mature miRNA sequence (termed miR). Both hairpin and mature sequences are available for searching and browsing, and entries can also be retrieved by name, keyword, references and annotation. All sequence and annotation data are also available for download.

The term“nucleotides”, as used herein, refers to structural components, or building blocks, of DNA and RNA. Nucleotides consist of a base (one of four chemicals: adenine, thymine, guanine, and cytosine) plus a molecule of sugar and one of phosphoric acid. The term “nucleosides” refers to glycosylamine consisting of a nucleobase (often referred to simply base) bound to a ribose or deoxyribose sugar. Examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine. Nucleosides can be phosphorylated by specific kinases in the cell on the sugar's primary alcohol group (-CH2-OH), producing nucleotides, which are the molecular building blocks of DNA and RNA.

The term“polynucleotide”, as used herein, means a molecule of at least 10 nucleotides and of not more than 45 nucleotides covalently linked together. Preferably, the polynucleotides of the present invention are molecules of 15 to 45 nucleotides or 10 to 35 nucleotides in length, more preferably of 16 to 28 nucleotides or 18 to 23 nucleotides in length, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides in length, not including optional spacer elements and/or elongation elements. The depiction of a single strand of a polynucleotide also defines the sequence of the complementary strand. Polynucleotides may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequences. The term“polynucleotide” means a polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and RNA molecules, both sense and anti-sense strands. In detail, the polynucleotide may be DNA, both cDNA and genomic DNA, RNA, cRNA or a hybrid, where the polynucleotide sequence may contain combinations of deoxyribonucleotide or ribonucleotide bases, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. Polynucleotides may be obtained by chemical synthesis methods or by recombinant methods.

In the context of the present invention, a polynucleotide as a single polynucleotide strand provides a probe (e.g. miRNA capture probe) that is capable of binding to, hybridizing with, or detecting a target of complementary sequence, such as a nucleotide sequence of a miRNA, through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. Polynucleotides in their function as probes may bind target sequences, such as nucleotide sequences of miRNAs, lacking complete complementarity with the polynucleotide sequences depending upon the stringency of the hybridization condition. There may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded polynucleotide described herein. However, if the number of mutations is so great that no hybridization can occur under even the least stringent hybridization conditions, the sequences are no complementary sequences. The polynucleotide variants including polynucleotide fragments or polynucleotide mutants and the miRNA variants including miRNA fragments or miRNA mutants are further defined below. Described herein are polynucleotides in form of single polynucleotide strands as probes for binding to, hybridizing with or detecting complementary sequences of miRNAs (targets) having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34.

The polynucleotide, e.g. the polynucleotide used as a probe for detecting a miRNA, may be unlabeled, directly labeled, or indirectly labeled, such as with biotin to which a streptavidin complex may later bind.

The term“differential expression” of a nucleic acid molecule, as used herein, refers to a qualitative and/or quantitative difference in the temporal and/or local nucleic acid molecule expression pattern, e.g. within blood. Thus, a differentially expressed nucleic acid molecule may qualitatively have its expression altered, including an activation or inactivation in, for example, blood from a untrained subject versus blood from a trained subject. The difference in nucleic acid molecule expression may also be quantitative, e.g. in that expression is modulated, i.e. either up-regulated, resulting in an increased amount of the nucleic acid molecule, or down- regulated, resulting in a decreased amount of the nucleic acid molecule. The degree to which nucleic acid molecule expression differs need only be large enough to be quantified via standard expression characterization techniques, e.g. by quantitative hybridization (e.g. to a microarray, to beads), amplification (PCR, RT-PCR, qRT-PCR, high-throughput RT-PCR), ELISA for quantitation, next generation sequencing (e.g. ABI SOLID, Illumina Genome Analyzer, Roche 454 GS FL), flow cytometry (e.g. LUMINEX) and the like.

The term“label”, as used herein, means a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and other entities which can be made detectable. A label may be incorporated into nucleic acids at any position, e.g. at the 3’ or 5’ end or internally. The polynucleotide for detecting a miRNA (polynucleotide probe) and/or the miRNA itself may be labeled. The term“stringent hybridization conditions”, as used herein, means conditions under which a first nucleic acid sequence (e.g. polynucleotide in its function as a probe for detecting a miRNA) will hybridize to a second nucleic acid sequence (e.g. target sequence such as nucleotide sequence of a miRNA), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5 to 10°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm may be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 20°C for short probes (e.g., about 10-35 nucleotides) and up to 60°C for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C; or 6x SSPE, 10 % formamide, 0.01 %, Tween 20, 0.1 x TE buffer, 0.5 mg/ml BSA, 0.1 mg/ml herring sperm DNA, incubating at 42°C with wash in 05x SSPE and 6x SSPE at 45°C.

The term “antisense”, as used herein, refers to a nucleotide sequence which is complementary to a specific DNA or RNA sequence. The term“antisense strand” is used in reference to a nucleic acid strand that is complementary to the“sense” strand.

Residues in two or more polynucleotide s are said to“correspond” to each other if the residues occupy an analogous position in the polynucleotide structures. It is well known in the art that analogous positions in two or more polynucleotides can be determined by aligning the polynucleotide sequences based on nucleic acid sequence or structural similarities. Such alignment tools are well known to the person skilled in the art and can be, for example, obtained on the World Wide Web, for example, ClustalW (see www.ebi.ac.uk/clustalw) or Align (see http://www.ebi.ac.uk/emboss/align/index.html) using standard settings, preferably for Align EMBOSS ::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

The term“level”, as used herein, refers to an amount (measured for example in grams, mole, or counts such as ion or fluorescence counts) or concentration (e.g. absolute or relative concentration) of the miRNA(s) described herein, in particular of the miRNA(s) having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34. The term“level”, as used herein, also comprises scaled, normalized, or scaled and normalized amounts or values. Preferably, the level determined herein is the expression level.

The term“AUC”, as used herein, relates to an abbreviation for the area under a curve. In particular, it refers to the area under a Receiver Operating Characteristic (ROC) curve. The term“Receiver Operating Characteristic (ROC) curve”, as used herein, refers to a plot of the true positive rate against the false positive rate for the different possible cut points of a diagnostic test. It shows the trade-off between sensitivity and specificity depending on the selected cut point (any increase in sensitivity will be accompanied by a decrease in specificity). The area under an ROC curve is a measure for the accuracy of a test (the larger the area the better, optimum is 1 , a random test would have a ROC curve lying on the diagonal with an area of 0.5 (see, for reference, for example, JP. Egan. Signal Detection Theory and ROC Analysis).

The term“fitness (or physical fitness)”, as used herein, means the state of health and well-being and, more specifically, the ability to perform aspects of sports, occupations and daily activities. It is further considered as a measure of the body’s ability to function efficiently and effectively in work and leisure activities, to be healthy, to resist hypokinetic diseases, and to meet emergency situations. Fitness (or physical fitness) is generally achieved through mo derate- vigorous physical exercise and sufficient rest. Components of fitness (or physical fitness) are: cardiorespiratory/cardiovascular endurance, muscular endurance, muscular strength, muscular power, flexibility, balance, speed, and body composition. Cardiorespiratory/cardiovascular endurance and muscular strength are preferably the components used to assess the fitness of a person. Also testing the other components, offers a more complete picture of overall fitness, along with health and athleticism. Fitness (or physical fitness) can, for example, be trained by jogging, running, walking, hiking, treadmill training, swimming, and/or cycling. The term “fitness (or physical fitness)” encompasses “cardiorespiratory/cardiovascular fitness”.

The term “cardiorespiratory/cardiovascular fitness”, as used herein, refers to the efficiency with which the heart pumps blood and oxygen through the body. Improving cardiorespiratory/cardiovascular fitness allows to keep running and exercise longer. It also has a number of health benefits: reducing the risk of heart disease and strokes, helping to lose weight, and reducing the risk of osteoporosis (weakening of bones). Cardiorespiratory/cardiovascular fitness can be measured using VC max, a measure of the amount of oxygen the body can uptake and utilize. Aerobic exercise, which improves cardiorespiratory fitness, involves movement that increases the heart rate to improve the body’s oxygen consumption. This form of exercise is an important part of all training regiments ranging from professional athletes to the everyday person.

The term“fitness (or physical fitness) level”, as used herein, means the body’s ability to withstand a physical workload (how much) and to recover in a timely manner. It refers to the fitness (or physical fitness) grade a person presently has. The fitness (or physical fitness) level of a person can increase, decrease, or remain stable. The fitness (or physical fitness) level of a person is the higher the more the person is able to withstand a physical workload and/or the faster the person is able to recover from the exercise.

Athletes, in particular amateur and professional athletes, benefit greatly from knowing their current fitness level and from monitoring their fitness level over time. Professional athletes are in training and it helps them to get the most efficient use of their exercise sessions. Also, knowing their fitness level helps them to plan their exercise strategy with the goal of a championship meet, match, run, etc. The same benefits apply to a regular person (amateur athlete) planning their daily/weekly exercise routine. Avoiding overtraining, undertraining and injuries is important to everyone.

The present inventors identified miRNAs which allow the determination and monitoring of the fitness level of a person in a cheap, simple, effective, and individualized way. The present inventors found, for example, that an increase of the level of the miRNAs having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID: 4 to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20 to SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 29 to SEQ ID NO: 33 over time is indicative for an improvement of the fitness of a person and that a decrease of the level of the miRNAs having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID: 4 to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20 to SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 29 to SEQ ID NO: 33 over time is indicative for a deterioration of the fitness of a person. In addition, the present inventors found, for example, that a decrease of the miRNAs having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID: 12 to SEQ ID NO: 19, SEQ ID NO: 23 to SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 34 over time is indicative for an improvement of the fitness of a person and that an increase of the miRNAs having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID: 12 to SEQ ID NO: 19, SEQ ID NO: 23 to SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 34 over time is indicative for an deterioration of the fitness of a person. The term“endurance training”, as used herein, refers to the act of exercising to increase endurance. The term endurance training generally refers to training the aerobic system as opposed to the anaerobic system. The need for endurance in sports is often predicated as the need of cardiovascular and simple muscular endurance, but the issue of endurance is far more complex. Endurance can be divided into two categories including: general endurance and specific endurance. It can be shown that endurance in sport is closely tied to the execution of skill and technique. A well-conditioned athlete can be defined as, the athlete who executes his or her technique consistently and effectively with the least effort. Heart rate monitoring is one method to assess fitness in endurance athletes. By comparing heart rate over time fitness gains can be observed when the heart rate decreases for running or cycling at a given speed. In cycling the effect of wind on the cyclists speed is difficult to subtract out and so many cyclists now use power meters built into their bicycles. The power meter allows the athlete to actually measure power output over a set duration or course and allows direct comparison of fitness progression.

The term“strength training”, as used herein, refers to a type of physical exercise specializing in the use of resistance to induce muscular contraction which builds the strength, anaerobic endurance, and size of skeletal muscles. When properly performed, strength training can provide significant functional benefits and improvement in overall health and well-being, including increased bone, muscle, tendon, and ligament strength and toughness, improved joint function, reduced potential for injury, increased bone density, increased metabolism, increased fitness and improved cardiac function. Training commonly uses the technique of progressively increasing the force output of the muscle through incremental weight increases and uses a variety of exercises and types of equipment to target specific muscle groups. Strength training is primarily an anaerobic activity, although some proponents have adapted it to provide the benefits of aerobic exercise through circuit training. Strength training is typically associated with the production of lactate, which is a limiting factor of exercise performance. Regular endurance exercise leads to adaptations in skeletal muscle which can prevent lactate levels from rising during strength training.

The present inventors identified miRNAs which allow the determination of the (predominant) training mode/type of training and the optimization of the personal training of a person in a cheap, simple, effective, and individualized way. On the basis of miRNA level determination, the person receives recommendations on the direction in which the training should be adapted (e.g. in the direction of endurance and/or strength training) to further improve overall fitness. The present inventors found, for example, that an increase of the level of the miRNAs having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32 above the reference level of reference subjects who performed strength training is indicative for a predominant endurance training so that the recommendation would/should be to optimize the training in the direction of strength training to improve the overall fitness of the person. In addition, the present inventors found, for example, that a decrease of the level of the miRNAs having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 34 below the reference level of reference subjects who performed strength training is indicative for a predominant endurance training so that the recommendation would/should be to optimize the training in the direction of strength training to improve the overall fitness of the person.

The term“subject”, as used herein, refers to any subject who wants to know her/his fitness level or fitness development over time. The term“subject” encompasses untrained and trained subjects. An untrained subject is preferably a person who has never done any sport before, or has not done any sport in the last 6 months, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 year(s). A trained subject is preferably a person who is conducting exercise training in regular or non regular time intervals, more preferably in regular time intervals, e.g. at least 1, 2, 3, 4, 5, 6 time(s) per week or 7 times per week, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 time(s) per month. A trained person may be a professional athlete or an amateur athlete. The terms“subject”, “person” or“individual” may be used interchangeable herein.

The term“reference subject”, as used herein, refers to a person of whom the training condition is known. It is known, for example, how much the reference subject trains, at which intensity and what. It is also known whether the reference subject is an endurance athlete or strength athlete. In particular, the fitness level of the reference subject is known. The reference subject may be trained or untrained.

The subject and the reference subject are preferably humans.

The term“professional athlete”, as used herein, refers to a subject playing or making sports on a professional level, in particular for a living. For a professional athlete, training is a full-time job. She/he has achieved top standing in her/his chosen field through years of training. A professional athlete is a person with natural talent, stamina, and competitive drive. A professional athlete has excellent reflexes and coordination and is well disciplined when it comes to rigorous practice and training. The term“amateur athlete”, as used herein, refers to a subject making sport on a regular or non-regular basis in order to improve the overall fitness and health status. For an amateur athlete, making sport is a hobby.

The subject referred to herein is preferably an amateur athlete (hobby athlete).

The term“blood sample”, as used herein, refers to any blood sample from a subject containing at least one miRNA selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34. The term“blood sample”, as used herein, encompasses whole blood or a blood fraction such as serum, plasma, or blood cells.

The term“reference blood sample”, as used herein, refers to any blood sample from a subject containing at least one miRNA selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34. The term“reference blood sample”, as used herein, encompasses whole blood or a blood fraction such as serum, plasma, or blood cells.

Preferably, the reference blood sample is from the same source than the blood sample of the subject to be tested, e.g. both are whole blood samples. More preferably, the reference blood sample and the blood sample are from subjects of the same sex and/or similar age.

The blood sample may be obtained from a subject prior to the initiation of the training, during the training, and/or after the training.

It is particularly preferred that the blood sample is whole blood. The whole blood sample may be taken from a subject by conventional blood collection techniques. In this case, the whole blood sample has a volume of between 0.01 and 20 ml, more preferably of between 0.1 and 10 ml, even more preferably of between 0.5 and 8 ml and most preferably of between 1 and 5 ml. The whole blood sample is preferably collected by means of a bloodspot technique, e.g. using a microsampling device such as Mitra microsampling device. This technique requires smaller sample volumes, typically 45-60 mΐ for humans or less. For example, the whole blood may be extracted from the individual via a finger prick with a needle or lancet. Thus, the whole blood sample may have the form of a blood drop. Said blood drop is then placed on an absorbent probe, e.g. a hydrophilic polymeric material such as cellulose, which is capable of absorbing the whole blood. Once sampling is complete, the blood spot is dried in air before transferring or mailing to labs for processing. Because the blood is dried, it is not considered hazardous. Thus no special precautions need be taken in handling or shipping. Once at the analysis site, the desired components, e.g. miRNAs, are extracted from the dried blood spots into a supernatant which is then further analyzed. This technique is suitable for monitoring the fitness level of subjects at home (on a home care/home sampling basis) or for screening purposes. Embodiments of the invention

Fitness training is primarily performed on the basis of universal exercise plans. Such a training is unindividualized and, thus, not always promising. The present inventors identified miRNAs which allow the determination and monitoring of the fitness level of a person in a cheap, simple and effective way. They also allow the determination of the training mode/type of training and the optimization of the personal training of the person. Thus, the present inventors developed a solution that is geared to the needs of people, it can be integrated into person’s daily working, studying or household schedules. It is specially tailored to and individually designed for a person. It ensures lasting wellbeing, and in particular, prevents cardiovascular or other common diseases.

Thus, in a first aspect, the present invention relates to a method of monitoring the fitness level of a subject comprising the steps of:

(i) determining the level of at least one miRNA (e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 miRNA(s), or 34 miRNAs) in a blood sample of a subject at a first point in time,

(iii) comparing said levels determined at the different time points,

The blood sample is isolated from the subject at a first point in time and at a later point in time. The comparison step allows the monitoring of the fitness level of the subject over time (longitudinal analysis).

Preferably, the time period between the first point in time and the later point(s) in time amounts to at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days (1 week), at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months (1 year). More preferably, the time period between the first point in time and the later point(s) in time amounts to between 1 month and 6 months, e.g. 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. Even more preferably, the time period between the first point in time and the later point(s) in time amounts to between 1 month and 3 months, e.g. 1 month, 2 months, or 3 months. For example, the subject may be routinely checked, e.g. 2, 3, or 4 times per year. The subject may be (re)tested at 2, 3, 4, 5, 6 7, 8, 9, or 10 time points (first point in time and further point(s) in time). The monitoring may be conducted over a time period of 6 months, 12 months (1 year), 2 years (24 months), 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years (in total) depending on the desired training period or monitoring period.

The type of blood sample at the first point in time and at the later point(s) in time is preferably the same. It is particularly preferred that the blood sample at the first point in time is a whole blood sample and the blood sample at the later point(s) in time is a whole blood sample.

In a preferred embodiment, the level of the at least one miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (hsa-miR-378a-3p,“up if fit”),

SEQ ID NO: 2, SEQ ID NO: 4 to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20 to SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 29 to SEQ ID NO: 33 which

(i) increases over time indicates that the fitness level of the subject improves,

(ii) does not change over time indicates that the fitness level of the subject is unaltered, or

(iii) decreases over time indicates that the fitness level of the subject deteriorates/worsens.

In another preferred (additional or alternative) embodiment, the level of the at least one miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 (hsa-miR-155-5p“down if fit”), SEQ ID NO: 9, SEQ ID NO: 12 to SEQ ID NO: 19, SEQ ID NO: 23 to SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 34 which

(i) decreases over time indicates that the fitness level of the subject improves,

(ii) does not change over time indicates that the fitness level of the subjects is unaltered, or

(iii) increases over time indicates that the fitness level of the subject deteriorates/worsens.

The detection of a decrease/an increase (dependent on the miRNA detected) of the level over time indicates that the fitness level improves/deteriorates in the subject. Preferably, said decrease/increase is at least 0.4-fold, at least 0.5-fold, at least 0.6-fold or at least 0.7-fold over time. More preferably, said decrease/increase is at least 0.8-fold or at least 0.9-fold over time. Even more preferably, said decrease/increase is at least 1.2-fold or at least 1.5-fold over time. Most preferably, said decrease/increase is at least 2.0-fold or at least 3.0-fold over time. For example, said decrease/increase may be determined over 6 months, 1 year (12 months) or over 2 years (24 months) depending on the desired training period or monitoring period.

A level which does not change over time indicates that the fitness level is unaltered, unchanged, or stable in the subject. In this respect, the term“does not change over time” preferably means that the level varies over time between 0 and < 20%, e.g. 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 19.9, 19.99, or 19.999%. The term“does not change over time” in this respect preferably further means that the detected level variation is within the accuracy of a measurement. The accuracy of a measurement depends on the measurement method used. The level may be constant over time.

If the level of more than one miRNA is determined, e.g. of at least two miRNAs, it is referred in steps (i) and (ii) of the method of monitoring the fitness level of a subject to the determination of the level of at least two miRNAs which are comprised in a set. In a more preferred embodiment, the level of the miRNAs comprised in one of the sets of Table 1 is determined in steps (i) and (ii).

Table 1: Preferred miRNA sets

In another more preferred embodiment, the level of the miRNAs comprised in one of the sets of Table 2 is determined in steps (i) and (ii).

Table 2: Preferred miRNA sets

(i) determining the level of at least one miRNA (e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 miRNA(s), or 34 miRNAs) in a blood sample of a subject, and

(ii) comparing the level of the at least one miRNA to a reference, in particular to a reference level,

The blood sample is isolated from the subject. The comparison step allows the determination of the fitness level of the subject.

The reference may represent a mathematical function, an algorithm, a classifier, or a numeric threshold determined from a reference level of a reference subject or from a plurality of reference levels derived from reference subjects. The reference subject is a person of whom the training condition is known. It is known, for example, how much the reference subject trains, at which intensity and what. It is also known whether the reference subject is an endurance athlete or strength athlete. In particular, the fitness level of the reference subject is known. The reference subject may be a trained or untrained.

Preferably, the reference is a reference level. The reference level may be a level determined by measuring at least one reference blood sample, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference blood sample(s), from at least one reference subject, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference subject(s). The reference subject has preferably a specific/defined fitness level so that the comparison allows the categorization of a subject to be tested in a specific training group/fitness level group. For example, the subject can be placed after the comparison step in a group that includes subjects who are fitter than the reference subject(s), in a group that includes subjects who are not so fit as the reference subject(s), or in a group that includes subjects who have a fitness level which is comparable with the fitness level of the reference subject(s).

The reference subject may be untrained or trained. In a preferred embodiment, the reference level is the level determined by measuring at least one reference blood sample, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference blood sample(s), from at least one untrained reference subject, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 untrained reference subject(s).

More preferably,

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (hsa-miR-378a-3p,“up if fit”), SEQ ID NO: 2, SEQ ID NO: 4 to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20 to SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 29 to SEQ ID NO: 33 above the reference level indicates that the subject has a fitness level which is better than the fitness level of an untrained reference subject, or

(ii) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 (hsa-miR-155-5p“down if fit”), SEQ ID NO: 9, SEQ ID NO: 12 to SEQ ID NO: 19, SEQ ID NO: 23 to SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 34 below the reference level indicates that the subject has a fitness level which is better than the fitness level of an untrained reference subject.

In another (additional or alternative) preferred embodiment, the reference level is the level determined by measuring at least one reference blood sample, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference blood sample(s), from at least one trained reference subject, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,

100, 150, 200, 250, 300, 400, 500, or 1.000 trained reference subject(s).

More preferably,

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (hsa-miR-378a-3p,“up if fit”), SEQ ID NO: 2, SEQ ID NO: 4 to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20 to SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 29 to SEQ ID NO: 33 comparable with or above the reference level indicates that the subject has a fitness level which is comparable with or better than the fitness level of a trained reference subject,

(ii) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (hsa-miR-378a-3p,“up if fit”), SEQ ID NO: 2, SEQ ID NO: 4 to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20 to SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 29 to SEQ ID NO: 33 below the reference level indicates that the subject has a fitness level which is worse than the fitness level of a trained reference subject,

(iii) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 (hsa-miR-155-5p“down if fit”), SEQ ID NO: 9, SEQ ID NO: 12 to SEQ ID NO: 19, SEQ ID NO: 23 to SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 34 comparable with or below the reference level indicates that the subject has a fitness level which is comparable with or better than the fitness level of a trained reference subject, or

(iv) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 (hsa-miR-155-5p“down if fit”), SEQ ID NO: 9, SEQ ID NO: 12 to SEQ ID NO: 19, SEQ ID NO: 23 to SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 34 above the reference level indicates that the subject has a fitness level which is worse than the fitness level of a trained reference subject. The detection of an up-regulation or a down-regulation (dependent on the miRNA detected) of the level compared to the reference level allows to determine the fitness level of the subject. In particular, the level of the at least one miRNA is at least 0.4-fold, at least 0.5- fold, at least 0.6-fold or at least 0.7-fold, preferably at least 0.8-fold or at least 0.9-fold, more preferably at least 1.2-fold or at least 1.5-fold, and even more preferably at least 2.0-fold or at least 3.0-fold below/above the reference level. For example, the level of the at least one miRNA is at least 0.4-fold, at least 0.5-fold, at least 0.6-fold, at least 0.7-fold, at least 0.8-fold, at least 0.9-fold, at least 1.0-fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2.0- fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, or at least 3.0-fold below/above the reference level.

It is practicable to take one reference blood sample per reference subject for analysis. If additional reference blood samples are required, e.g. to determine the reference level in different reference blood samples, the same reference subject may be (re)tested.

Said reference level may be an average reference level. It may be determined by measuring reference levels and calculating the“average” value (e.g. mean, median or modal value) thereof. It is preferred that the reference blood sample is from the same source than the blood sample isolated from the subject to be tested, e.g. a whole blood sample. It is further preferred that the reference level is obtained from a reference subject of the same gender (e.g. female or male) and/or of a similar age/phase of life (e.g. adults or elderly) than the subject to be tested or analysed.

If the level of more than one miRNA is determined, e.g. of at least two miRNAs, it is referred in step (i) of the method of determining the fitness level of a subject to the determination of the level of at least two miRNAs which are comprised in a set. In a more preferred embodiment, the level of the miRNAs comprised in one of the sets of Table 1 is determined in step (i).In another more preferred embodiment, the level of the miRNAs comprised in one of the sets of Table 2 is determined in step (i).

(i) determining the level of at least one miRNA (e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 miRNA(s), or 29 miRNAs) in a blood sample of a subject, and

The blood sample is isolated from the subject. The comparison step allows the determination of the (predominant) training mode/type of training of a subject. Preferably, the (predominant) training mode/type of training is endurance or strength training. In particular, it is possible with the above method to determine whether the subjects (predominantly) performs endurance or strength training.

It is alternatively possible to classify/categorize the subject in the group of endurance or strength athletes by (i) determining the level of at least one miRNA in a blood sample of a subject, and (ii) comparing the level of the at least one miRNA to a reference level, wherein the at least one miRNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 30, and SEQ ID NO: 32 to SEQ ID NO: 34.

It is also possible in an alternative approach to distinguish endurance from strength athletes or to distinguish between endurance and strength athletes by (i) determining the level of at least one miRNA in a blood sample of a subject, and (ii) comparing the level of the at least one miRNA to a reference level, wherein the at least one miRNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 30, and SEQ ID NO: 32 to SEQ

ID NO: 34.

In the method of determining the (predominant) training mode/type of training of a subject, it is preferred that the reference level is the level determined by measuring at least one reference blood sample, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,

19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,

44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference blood sample(s), from at least one reference subject who (predominantly) performed strength training (strength athlete), e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,

22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,

47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference subject(s) who performed (predominantly) strength training (strength athlete(s)).

More preferably,

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 (hsa-miR-484“up if endurance”), SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32 above the reference level indicates that the subject makes (predominantly) endurance training, or

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (hsa-miR-378a-3p,“down if endurance”), SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 34 below the reference level indicates that the subject makes (predominantly) endurance training. It is also (alternatively or additionally) preferred that the reference level is the level determined by measuring at least one reference blood sample, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8,

9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,

35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference blood sample(s), from at least one reference subject who performed (predominantly) endurance training (endurance athlete), e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8,

35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference subject(s) who performed (predominantly) endurance training (endurance athlete(s)).

More preferably,

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 (hsa-miR-484“down if strength”), SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32 below the reference level indicates that the subject makes (predominantly) strength training, or

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (hsa-miR-378a-3p,“up if strength”), SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 34 above the reference level indicates that the subject makes (predominantly) strength training. The detection of an up-regulation or a down-regulation (dependent on the miRNA detected) of the level compared to the reference level allows to determine whether the subject makes/performs (predominantly) strength or endurance training. In particular, the level of the at least one miRNA is at least 0.4-fold, at least 0.5-fold, at least 0.6-fold or at least 0.7-fold, preferably at least 0.8-fold or at least 0.9-fold, more preferably at least 1.2-fold or at least 1.5- fold, and even more preferably at least 2.0-fold or at least 3.0-fold below/above the reference level. For example, the level of the at least one miRNA is at least 0.4-fold, at least 0.5-fold, at least 0.6-fold, at least 0.7-fold, at least 0.8-fold, at least 0.9-fold, at least 1.0-fold, at least 1.1- fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2.0-fold, at least 2.1-fold, at least 2.2- fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, or at least 3.0-fold below/above the reference level.

If the level of more than one miRNA is determined, e.g. of at least two miRNAs, it is referred in step (i) of the method of determining the (predominant) training mode/type of training of a subject to the determination of the level of at least two miRNAs which are comprised in a set. In a more preferred embodiment, the level of the miRNAs comprised in one of the sets of Table 3 is determined in step (i).

Table 3: Preferred miRNA sets

In another more preferred embodiment, the level of the miRNAs comprised in one of the sets of Table 4 is determined in step (i).

Table 4: Preferred miRNA sets

(ii) comparing the level of the at least one miRNA to a reference level,

The blood sample is isolated from the subject. Preferably, the optimizing encompasses the orientation of the training in the direction of endurance or strength training.

In a preferred embodiment, the reference level is the level determined by measuring at least one reference blood sample, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference blood sample(s), from at least one reference subject who performed strength training (strength athlete), e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,

22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46,

47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference subject(s) who performed strength training (strength athlete(s)).

More preferably,

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 (hsa-miR-484“up if endurance”), SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32 above the reference level indicates that the subject (makes (predominantly) endurance training and) should/could optimize the training in the direction of strength training, or (i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (hsa-miR-378a-3p,“down if endurance”), SEQ ID NO: 9, SEQ ID

NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 34 below the reference level indicates that the subject (makes (predominantly) endurance training and) should/could optimize the training in the direction of strength training.

In another preferred embodiment, the reference level is the level determined by measuring at least one reference blood sample, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference blood sample(s), from at least one reference subject who performed endurance training (endurance athlete), e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 400, 500, or 1.000 reference subject(s) who performed endurance training (endurance athlete(s)).

More preferably,

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 (hsa-miR-484“down if strength”), SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32 below the reference level indicates that the subject (makes (predominantly) strength training and) should/could optimize the training in the direction of endurance training, or

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (hsa-miR-378a-3p,“up if strength”), SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 21 , SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 34 above the reference level indicates that the subject (makes (predominantly) strength training and) should/could optimize the training in the direction of endurance training.

The subject should/could optimize the training in the direction of endurance or strength training in order to further improve her/his fitness and to reach her/his training goals. In particular, the outcome of the above mentioned method is a suggestion/recommendation for the subject to adjust the training in order to (further) improve overall fitness and to reach specific training goals.

The detection of an up-regulation or a down-regulation (dependent on the miRNA detected) of the level compared to the reference level allows to determine whether the subject should/could optimize the training in the direction of endurance or strength training, in particular in order to improve her/his fitness and to reach her/his training goals. In particular, the level of the at least one miRNA is at least 0.4-fold, at least 0.5-fold, at least 0.6-fold or at least 0.7-fold, preferably at least 0.8-fold or at least 0.9-fold, more preferably at least 1.2-fold or at least 1.5-fold, and even more preferably at least 2.0-fold or at least 3.0-fold below/above the reference level. For example, the level of the at least one miRNA is at least 0.4-fold, at least 0.5-fold, at least 0.6-fold, at least 0.7-fold, at least 0.8-fold, at least 0.9-fold, at least 1.0-fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6- fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2.0-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7- fold, at least 2.8-fold, at least 2.9-fold, or at least 3.0-fold below/above the reference level.

If the level of more than one miRNA is determined, e.g. of at least two miRNAs, it is referred in step (i) of the method of optimizing personal training of a subject to the determination of the level of at least two miRNAs which are comprised in a set. In a more preferred embodiment, the level of the miRNAs comprised in one of the sets of Table 3 is determined in step (i).In another more preferred embodiment, the level of the miRNAs comprised in one of the sets of Table 4 is determined in step (i).

The determination of the level of the at least one miRNA may be carried out in the methods of the first to fourth aspect of the present invention by any convenient means for determining the level of a nucleotide sequence such as miRNA. For this purpose, qualitative, semi-quantitative and quantitative detection methods can be used. Quantitative detection methods are preferred. A variety of techniques are well known to the person skilled in the art. However, in the methods of the first to fourth aspect of the present invention, it is preferred that the level is determined by nucleic acid hybridization, nucleic acid amplification, polymerase extension, sequencing, mass spectroscopy, an immunochemical method or any combination thereof.

Preferably,

(i) the nucleic acid hybridization is performed using a microarray/biochip, or using in situ hybridization,

(ii) the nucleic acid amplification is performed using real-time PCR (RT-PCR) or quantitative real-time PCR (RT-qPCR),

(iii) the sequencing is next generation sequencing, or

(iv) the immunochemical method is an enzyme linked immunosorbent assay (ELISA).

Nucleic acid amplification, for example, may be performed using real time polymerase chain reaction (RT-PCR) such as real time quantitative PCR (RT-qPCR). The real time polymerase chain reaction (RT-PCR) may include the following steps: (i) extracting total RNA from the blood sample isolated from the subject, (ii) obtaining cDNA samples by RNA reverse transcription (RT) reaction using miRNA-specific primers, (iii) designing miRNA-specific cDNA forward primers and providing universal reverse primers to amplify the cDNA via polymerase chain reaction (PCR), (iv) adding a fluorescent probe to conduct PCR, and (v) detecting and comparing the variation in levels of miRNAs in the blood sample isolated from the subject relative to those of miRNAs in a reference blood sample isolated from a reference subject.

A variety of kits and protocols to determine the miRNA level by real time polymerase chain reaction (RT-PCR) such as real time quantitative PCR (RT qPCR) are available. For example, reverse transcription of miRNAs may be performed using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) according to manufacturer’s recommendations.

Nucleic acid hybridization, for example, may be performed using a microarray/biochip or in situ hybridization. For nucleic acid hybridization, for example, the polynucleotides (probes) described herein with complementarity to the corresponding miRNAs to be detected are attached to a solid phase to generate a microarray/biochip. Said microarray/biochip is then incubated with miRNAs, isolated (e.g. extracted) from the blood sample, which may be labelled or unlabelled. Upon hybridization of the labelled miRNAs to the complementary polynucleotide sequences on the microarray/biochip, the success of hybridisation may be controlled and the intensity of hybridization may be determined via the hybridisation signal of the label in order to determine the level of each tested miRNA in said blood sample.

Alternatively, the miRNA level may be determined using an immunochemical method, e.g. using an ELISA. Said method may include the following steps: (i) isolating miRNAs from a blood sample, (ii) hybridizing polynucleotide probes (complementary) to the miRNAs to obtain hybrids of said polynucleotides probes and said miRNAs, and (iii) binding said hybrids to antibodies capable of specifically binding hybrids of said polynucleotide probes and said miRNAs, and (iv) detecting the antibody-bound hybrids.

In the methods of the first to fourth aspect of the present invention, it is preferred that the blood sample is a whole blood sample. The whole blood sample is preferably collected by means of a bloodspot technique, e.g. using a microsampling device such as Mitra microsampling device. For example, the whole blood may be extracted from the subject to be tested via a finger prick with a needle or lancet. Thus, the whole blood sample may have the form of a blood drop. Said blood drop is then placed on an absorbent probe, e.g. a hydrophilic polymeric material such as cellulose, which is capable of absorbing the whole blood. Once sampling is complete, the blood spot is dried in air before transferring or mailing to labs for processing. Because the blood is dried, it is not considered hazardous. Thus no special precautions need be taken in handling or shipping. Once at the analysis site, the miRNAs are extracted from the dried blood spots into a supernatant which is then further analyzed. This technique is suitable for monitoring the fitness level of subjects, determining the fitness level of subjects, determining the (predominant) training mode/type of training of a subject, or optimizing personal training of a subject at home (on a home care/home sampling basis) or for screening purposes. Alternatively, a card of an adsorbent probe, e.g. a hydrophilic polymeric material such as cellulose, which is capable of absorbing the whole blood, may be used for blood collection/uptake. The blood is spotted onto the card of an absorbent probe and subsequently dried (dry blood spot (DBS)). This card can then be shipped and the absorbed material, in particular the miRNA comprised therein, can be further analyzed.

The subject may be an amateur athlete or a professional athlete. Preferably, the subject is an amateur athlete, i.e. no professional athlete.

In the methods of the first to fourth aspect of the present invention, it is further preferred that the level of the at least one miRNA is the expression level of said at least one miRNA.

The methods of the first to fourth aspect of the present invention are further in vitro methods.

In a fifth aspect, the present invention relates to the use of at least one polynucleotide (probe/primer, in particular primer pair) for detecting at least one miRNA (e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,

31, 32, 33 miRNA(s), or 34 miRNAs) in a blood sample isolated from/of a subject for monitoring the fitness level of the subject, wherein the at least one miRNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34 and a sequence having at least 90%, preferably at least 95%, more preferably at least 99%, i.e. at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto.

In a preferred embodiment,

(i) the at least one polynucleotide is at least partially (reverse) complementary, preferably (reverse) complementary, to the at least one miRNA mentioned above, or

(ii) the at least one polynucleotide has at least 90%, preferably at least 95%, more preferably at least 99%, i.e. at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the polynucleotide according to (i).

It is particularly preferred that the polynucleotide as defined in (ii) has at least 90%, preferably at least 95%, more preferably at least 99%, i.e. at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity over a continuous stretch of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 ,44, or more nucleotides, preferably over the whole length, to the polynucleotide according to

(i)·

In addition, the polynucleotide as defined in (ii) (i.e. polynucleotide variant) is only regarded as a polynucleotide as defined in (ii) (i.e. polynucleotide variant) within the context of the present invention, if it is still capable of binding to, hybridizing with, or detecting the respective target nucleic acid molecule, i.e. the target nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34, through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation under stringent hybridization conditions. The skilled person can readily assess whether a polynucleotide as defined in (ii) (i.e. polynucleotide variant) is still capable of binding to, hybridizing with, recognizing or detecting the respective target nucleic acid molecule, i.e. the target nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34. Suitable assays to determine whether hybridization under stringent conditions still occurs are well known in the art. However, as an example, a suitable assay to determine whether hybridization still occurs comprises the steps of: (a) incubating the polynucleotide as defined in (ii) or (iii) attached onto a biochip with the respective target nucleic acid molecule, i.e. the target nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34, (b) washing the biochip to remove unspecific bindings, (c) subjecting the biochip to a detection system, and (d) analyzing whether the polynucleotide can still hybridize with the respective target nucleic acid molecule. As a positive control, the respective non-mutated polynucleotide as defined in (i) may be used. Preferably stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C; or 6x SSPE, 10 % formamide, 0.01 %, Tween 20, 0.1 x TE buffer, 0.5 mg/ml BSA, 0.1 mg/ml herring sperm DNA, incubating at 42°C with wash in 05x SSPE and 6x SSPE at 45°C.

The at least one miRNA is detected by the at least one polynucleotide in a blood sample. The blood sample is preferably a whole blood sample.

The at least one polynucleotide (probe/primer, in particular primer pair) described above is useful for conducting the method of the first aspect of the present invention.

In a sixth aspect, the present invention relates to the use of at least one polynucleotide (probe/primer, in particular primer pair) for detecting at least one miRNA (e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,

31, 32, 33 miRNA(s), or 34 miRNAs) in a blood sample isolated from/of a subject for determining the fitness level of the subject,

wherein the at least one miRNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34 and a sequence having at least 90%, preferably at least 95%, more preferably at least 99%, i.e. at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto.

In a preferred embodiment,

It is particularly preferred that the polynucleotide as defined in (ii) has at least 90%, preferably at least 95%, more preferably at least 99%, i.e. at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity over a continuous stretch of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 ,44, or more nucleotides, preferably over the whole length, to the polynucleotide according to (ⁱr As to the polynucleotide variants, it is referred to the fifth aspect of the present invention.

The at least one polynucleotide (probe/primer, in particular primer pair) described above is useful for conducting the method of the second aspect of the present invention.

In a seventh aspect, the present invention relates to the use of at least one polynucleotide (probe/primer, in particular primer pair) for detecting at least one miRNA (e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 miRNA(s), or 29 miRNAs) in a blood sample isolated from/of a subject for determining the training mode/type of training of the subject,

wherein the at least one miRNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 30, and SEQ ID NO: 32 to SEQ ID NO: 34 and a sequence having at least 90%, preferably at least 95%, more preferably at least 99%, i.e. at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto.

In a preferred embodiment,

(i)·

As to the polynucleotide variants, it is referred to the fifth aspect of the present invention.

The at least one miRNA is detected by the at least one polynucleotide in a blood sample. The blood sample is preferably a whole blood sample. The subject may be an amateur athlete or a professional athlete. Preferably, the subject is an amateur athlete, i.e. no professional athlete.

The at least one polynucleotide (probe/primer, in particular primer pair) described above is useful for conducting the method of the third aspect of the present invention.

In an eight aspect, the present invention relates to the use of least one polynucleotide (probe/primer, in particular primer pair) for detecting at least one miRNA (e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 miRNA(s), or 29 miRNAs) in a blood sample isolated ffom/of a subject for optimizing personal training of the subject,

In a preferred embodiment,

(i)·

The at least one polynucleotide (probe/primer, in particular primer pair) described above is useful for conducting the method of the fourth aspect of the present invention. In a ninth aspect, the present invention relates to a kit for monitoring the fitness level or determining the fitness level of a subject comprising:

(i) means for determining the level of at least one miRNA (e.g. at least 1, 2, 3, 4, 5, 6, 7, 8,

9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,

33 miRNA(s), or 34 miRNAs) in a blood sample of/isolated from a subject, and (ii) optionally at least one reference,

It is preferred that the means in (i) comprise

at least one polynucleotide (probe), in particular according to the fifth or sixth aspect of the present invention,

at least one primer pair, in particular according to the fifth or sixth aspect of the present invention, and/or

at least one polynucleotide (probe), in particular according to the fifth or sixth aspect of the present invention, and at least one antibody capable of binding a hybrid of said at least one polynucleotide (probe) and said at least one miRNA.

Said means allow to determine the level of the at least one miRNA in a blood sample ofisolated from a subject and, thus, to monitored or determine the fitness level of a subject.

The at least one polynucleotide (probe) may be part of a microarray/biochip or may be attached to beads of a beads-based multiplex system.

The at least one polynucleotide (primer, primer pair) may be part of a RT-PCR system, a PCR-system, or a next generation sequencing system.

Said means may further comprise a microarray, a RT-PCT system, a PCR-system, a flow cytometer, a Luminex system and/or a next generation sequencing system.

It is preferred that the reference in (ii) is the reference level. As to the reference and/or reference level, it is referred to the first and second aspect of the present invention.

The blood sample is preferably a whole blood sample. The subject may be an amateur athlete or a professional athlete. Preferably, the subject is an amateur athlete, i.e. no professional athlete.

It is further preferred that the kit is useful for conducting the methods of the first and/or second aspect. It is also preferred that the kit comprises instructions on how to carry out the methods of the first and/or second aspect.

In a tenth aspect, the present invention relates to a kit for determining the training mode/type of training or optimizing personal training of a subject comprising: (i) means for determining the level of at least one miRNA (e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 miRNA(s), or 29 miRNAs) in a blood sample of/isolated from a subject, and

(ii) optionally at least one reference,

It is preferred that the means in (i) comprise

at least one polynucleotide (probe), in particular according to the seventh or eighth aspect of the present invention,

at least one primer pair, in particular according to the seventh or eighth aspect of the present invention, and/or

at least one polynucleotide (probe), in particular according to the seventh or eighth aspect of the present invention, and at least one antibody capable of binding a hybrid of said at least one polynucleotide (probe) and said at least one miRNA.

Said means allow to determine the level of the at least one miRNA in a blood sample of/isolated from a subject and, thus, to determine the training mode/type of training or to optimize personal training of a subject.

It is preferred that the reference in (ii) is the reference level. As to the reference and/or reference level, it is referred to the third and fourth aspect of the present invention.

It is further preferred that the kit is useful for conducting the methods of the third and/or fourth aspect. It is also preferred that the kit comprises instructions on how to carry out the methods of the third and/or fourth aspect.

The kit of the ninth or tenth aspect of the present invention may further comprise

(iii) a container, and/or (iv) a data carrier.

The data carrier may be a non-electronical data carrier, e.g. a graphical data carrier such as an information leaflet, an information sheet, a bar code or an access code, or an electronical data carrier such as a floppy disk, a compact disk (CD), a digital versatile disk (DVD), a microchip or another semiconductor-based electronical data carrier. The access code may allow the access to a database, e.g. an internet database, a centralized, or a decentralized database. The access code may also allow access to an application software that causes a computer to perform tasks for computer users or a mobile app which is a software designed to run on smartphones and other mobile devices.

Said data carrier may further comprise the at least one reference, e.g. the reference level of the level of the at least one miRNA determined herein. In case that the data carrier comprises an access code which allows the access to a database, said at least one reference, e.g. said reference level may be deposited in this database.

The data carrier may also comprise information or instructions on how to carry out the methods of the first to fourth aspect of the present invention.

Said kit may also comprise materials desirable from a commercial and user standpoint including a buffer(s), a reagent(s) and/or a diluent(s) for determining the level mentioned above.

(i) a microsampling device,

(ii) optionally an access code, and

(iii) optionally instructions on how to use the kit.

Microsampling devices allow the subject to self-collect an accurate blood specimen anywhere, anytime, with minimal instructions. Microsampling devices are known to the person skilled in the art. They may have a clamshell or cartridge format. Microsampling devices may comprise an absorbent polymeric tip designed to take up a fixed volume of blood by capillary action. The tip is attached to a handle by a plastic pin. Microsampling devices allow the extraction of whole blood from the subject via a finger prick with a needle or lancet. Thus, the whole blood sample may have the form of a blood drop. Said blood drop is then placed on an absorbent probe, e.g. a hydrophilic polymeric material such as cellulose, which is capable of absorbing the whole blood. Once sampling is complete, the blood spot is dried in air before transferring or mailing to labs for processing. Because the blood is dried, it is not considered hazardous. Thus no special precautions need be taken in handling or shipping. Once at the analysis site, the miRNAs are extracted from the dried blood spots into a supernatant which is then further analyzed. One example of a microsampling device is the Mitra microsampling device.

Instead of a microsampling device, a card of an adsorbent probe, e.g. a hydrophilic polymeric material such as cellulose, which is capable of absorbing the whole blood, may alternatively be used. The blood drop is placed on the card of an adsorbent probe and subsequently dried. This card is also designated as dried blood spot (DBS) card. Dried blood spot (DBS) analysis is an easy way of collecting, shipping and storing blood samples. In recent years the usage of dried blood spots (DBS) has gained increasing importance since this method shows strong advantages compared to the conventional collection and analysis of blood or plasma samples. These advantages include the need for remarkably lower blood volumes and easier shipping and storage, often at ambient temperatures. This leads to a simplification of the blood collection process and a significant reduction of the costs involved. The blood sample needs to be extracted from the DBS card prior to the analysis.

Preferably, the access code and/or the instructions on how to use the kit are comprised on a data carrier. The data carrier may be a non-electronical data carrier, e.g. a graphical data carrier such as an information leaflet, an information sheet, a bar code or an access code, or an electronical data carrier such as a floppy disk, a compact disk (CD), a digital versatile disk (DVD), a microchip or another semiconductor-based electronical data carrier. The access code may allow the access to a database, e.g. an internet database, a centralized, or a decentralized database. The access code may also allow access to an application software that causes a computer to perform tasks for computer users or a mobile app which is a software designed to run on smartphones and other mobile devices.

In a further aspect, the present invention relates to a method of classifying/categorizing a subject into the group of endurance athletes or strength athletes comprising the steps of:

(i) determining the level of at least one miRNA in a blood sample of a subject, and

(ii) comparing the level of the at least one miRNA to a reference level,

In a preferred embodiment, the reference level is the level determined by measuring at least one reference blood sample from at least one reference subject who performed strength training (strength athlete).

Preferably, (i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 (hsa-miR-484“up if endurance”), SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32 above the reference level indicates that the subject falls into the group of endurance athletes, or

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (hsa-miR-378a-3p,“down if endurance”), SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 34 below the reference level indicates that the subject falls into the group of endurance athletes. In another embodiment, the reference level is the level determined by measuring at least one reference blood sample from at least one reference subject who performed endurance training (endurance athlete).

Preferably,

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 (hsa-miR-484“down if strength”), SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32 below the reference level indicates that the subject falls into the group of strength athletes, or

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (hsa-miR-378a-3p,“up if strength”), SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 34 above the reference level indicates that the subject falls into the group of strength athletes.

In particular, the level of the at least one miRNA is at least 0.4-fold, at least 0.5-fold, at least 0.6-fold or at least 0.7-fold, preferably at least 0.8-fold or at least 0.9-fold, more preferably at least 1.2-fold or at least 1.5-fold, and even more preferably at least 2.0-fold or at least 3.0- fold below/above the reference level. For example, the level of the at least one miRNA is at least 0.4-fold, at least 0.5-fold, at least 0.6-fold, at least 0.7-fold, at least 0.8-fold, at least 0.9- fold, at least 1.0-fold, at least 1.1 -fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2.0- fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, or at least 3.0-fold below/above the reference level.

In a further aspect, the present invention relates to a method of distinguishing endurance from strength athletes or distinguishing between endurance and strength athletes comprising the steps of:

(ii) comparing the level of the at least one miRNA to a reference level,

Preferably,

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 (hsa-miR-484“up if endurance”), SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32 above the reference level indicates that the subject is an endurance athlete, or

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (hsa-miR-378a-3p,“down if endurance”), SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 34 below the reference level indicates that the subject is an endurance athlete.

In another embodiment, the reference level is the level determined by measuring at least one reference blood sample from at least one reference subject who performed endurance training (endurance athlete).

Preferably,

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 (hsa-miR-484“down if strength”), SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32 below the reference level indicates that the subject is a strength athlete, or

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (hsa-miR-378a-3p,“up if strength”), SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 21 , SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 34 above the reference level indicates that the subject is a strength athlete.

In a further aspect, the present invention relates to a method of determining the (best/most effective) training mode/type of training for a subject comprising the steps of:

(i) determining the expression profile of at least one miRNA in a blood sample of a subject, and

(ii) comparing the expression profile of the at least one miRNA to a reference expression profile,

The comparison allows to determine the (best/most effective) training mode/type of training for the subject, in particular in order to increase/improve the subject’s fitness level. Preferably, the comparison allows to determine whether endurance or strength training is the best/most effective training mode/type of training for the subject. The reference expression profile is preferably a profile determined by measuring at least one reference blood sample from at least one reference subject from whom it is known which training mode/type of training was the most effective one, e.g. endurance or strength training. This reference profile is preferably the profile determined from the subject before the training (originally) started. Preferably, the reference expression profile is a mean reference expression profile determined from a plurality of reference subjects.

Is the expression profile of the subject comparable (or even identical) with the reference expression profile of the reference subject, the trainings mode/type of training of the reference subject is also the best/most effective training mode/type for the subject. For example, is the expression profile of the subject comparable (or even identical) with the reference expression profile of the reference subject from whom it is known that endurance training was the most effective one, in particular in order to increase/improve reference subject’s fitness level, endurance training is also the best/most effective training mode/type of training for the subject. In addition, is the expression profile of the subject comparable (or even identical) with the reference expression profile of the reference subject from whom it is known that strength training was the most effective one, in particular in order to increase/improve reference subject’s fitness level, strength training is also the best/most effective training mode/type of training for the subject.

In a further aspect, the present invention relates to a method of determining the (best/most effective) form of nutrition for a subject, in particular in order to increase/improve the subject’s fitness level, comprising the steps of:

The comparison allows to determine the (best/most effective) form of nutrition for the subject, in particular in order to increase/improve the subject’s fitness level. Preferably, the comparison allows to determine whether carbohydrate rich nutrition/diet is the best/most effective form of nutrition for the subject. Alternatively, the comparison allows to determine whether protein rich nutrition/diet is the best/most effective form of nutrition for the subject. The reference expression profile is preferably a profile determined by measuring at least one reference blood sample from at least one reference subject from whom it is known which form of nutrition was the most effective one, in particular in order to increase/improve the subject’s fitness level. This reference profile is preferably the profile determined from the subject before the subject (originally) switched the nutrition/diet to the best/most effective one, e.g. carbohydrate or protein rich nutrition/diet. Preferably, the reference expression profile is a mean reference expression profile determined from a plurality of reference subjects.

Is the expression profile of the subject comparable (or even identical) with the reference expression profile of the reference subject, the form of nutrition of the reference subject is also the best/most effective form of nutrition for the subject, in particular in order to increase/improve the subject’s fitness level. For example, is the expression profile of the subject comparable (or even identical) with the reference expression profile of the reference subject from whom it is known that carbohydrate rich nutrition/diet was the most effective one, in particular in order to increase/improve reference subject’s fitness level, carbohydrate nutrition/diet is also the best/most effective form of nutrition for the subject. In addition, is the expression profile of the subject comparable (or even identical) with the reference expression profile of the reference subject from whom it is known that protein rich nutrition/diet was the most effective one, in particular in order to increase/improve reference subject’s fitness level, protein rich nutrition/diet is also the best/most effective form of nutrition for the subject.

As to the preferred embodiment, e.g. with respect to the specific blood sample type used, the reference subjects, the reference levels, the forms of expression profile/level determination, it is referred to the first to fourth aspect of the present invention and to the definition section. As to the preferred miRNA combinations, it is also referred to the miRNA sets disclosed in Tables 1 to 4.

The methods described above, in particular the methods of the first to fourth aspect of the present invention, are preferably carried out using artificial intelligence. Artificial intelligence allows the evaluation of digitally available information with the help of computers in order to make meaningful proposals, statements, or predictions.

Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope of invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art in the relevant fields are intended to be covered by the present invention.

BRIEF DESCRIPTION OF THE FIGURES The following Figures are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way.

Figure 1: miRNAs described herein with sequence identifier (SEQ ID NO:) and nucleotide sequence.

Figure 2: Table describing the molecular changes/miRNA deregulation between fit and unfit individuals. The first column lists the SEQ ID, the second the according miRNA name. Then median values and standard deviation for the two groups are provided (columns 3, 4, 5, and 6). Next, the fold change between the two groups (column 7), the raw and adjusted p-value of a t-test (columns 8 and 9) and the AUC is given (column 10). Column 11 contains the description of the miRNA.

Figure 3: Table describing the molecular changes/miRNA deregulation between strength and endurance athletes. The first column lists the SEQ ID, the second the according miRNA name. Then median values and standard deviation for the two groups are provided (columns 3,4,5, and 6). Next, the fold change between the two groups (column 7), the raw and adjusted p-value of a t-test (columns 8 and 9) and the AUC is given (column 10). Column 1 1 contains the description of the miRNA.

Figure 4: Example of an individual profile. For one test person that belongs to the least fit part of the cohort the miRNA values for seven important markers (top 7 of miRNAs listed in Figure 2, SEQ ID NO: 1, 2, 3, 4, 5, 6, and7) are provided in an intuitive way. The more RIGHT the values are the better. The miRNA values are normalized to a percent scale (in the peer cohort of all study participants) to show them in their context. Each testing round is marked by one triangle. The fitness of the test person decreased over the study. By using other machine learning or artificial intelligence applications the representation can be further condensed such that only one score is required.

EXAMPLES

The examples given below are for illustrative purposes only and do not limit the invention described above in any way.

Study design

The test persons have been recruited personally on public events (fares and similar). All participants gave written and informed consent on the study participation. Personal data such as the address (required for shipping the home sampling kits), general demographic information and relevant information with respect to the current fitness condition (body mass index, personal fitness estimation, number of training sessions per week, sports injuries) were collected. Further, test persons got a set of 100 training schedules personalized for their needs (e.g. tailored for light endurance or heavy strength training). One part of the cohort used the provided training schedules, the other part followed own training schedules. Altogether, almost 25,000 workouts have been performed by the test persons in the course of the study. The blood collection has been carried out at four distinct time intervals. Test persons had only two weeks to return their sample at each time point. The two weeks started at time points during the summer, autumn, winter, and spring, respectively.

RNA Extraction and Quality Control

For RNA extraction from micro-sampling devices two 10 mΐ Mitra tips (Neoteryx, California, USA) filled with whole blood were used as input. Extraction of total RNA incl. miRNA was performed by using the miRNeasy Serum/Plasma kit (Qiagen, Hilden Germany). For quantification and RNA quality control of purified samples the Small RNA Kit (Agilent Technologies, Santa Clara, USA) and Agilent 2100 Bioanalyzer instrument was used according to manufacturer’s instructions. qRT-PCR Analysis

Quantification of miRNA levels of Mitra samples was performed on the Fluidigm Biomark HD system using the 96.96 IFC controller and dynamic arrays according to manufacturer’s instructions (Fluidigm, California, USA). For normalization and inter-plate comparison one exogenous (cel-miR-39) and four endogenous RNA controls (RNU44, RNU48, RNU6b, RPL21) as well as a Mitra standard pooled control sample were measured on each dynamic array. Each miRNA and each control was measured in triplicates. Reverse Transcription, pre amplification and qRT-PCR reaction were carried out according to the manufacturer’s protocols (Fluidigm, California, USA).

Bioinformatics

All computations have been carried out with the freely available programming environment R. As statistical tests, both, t-test and Wilcoxon Mann- Whitney test were computed to find differentially regulated miRNAs between groups (e.g. between FIT and UNFIT or between ENDURANCE and STRENGTH). Since the test results between the two tests were concordant, only t-test results were reported. Besides the p-value, other metrics for accessing the quality were added, these include the fold change or the Area Under The Receiver Operator Characteristic Curve (AUC value). The p-values that have been computed were adjusted for multiple testing using the Benj amini-Hochberg approach, controlling the False Discovery rate. To present concise visualizations for users, different approaches have been developed. First, per miRNA and per Patient one bar is presented. For each measurement round of this miRNA in the patient on triangle is added labeled by the corresponding test round. miRNAs are adjusted in a manner that the rightmost position in the bar would correspond to the best fitness value and the leftmost position to the least fit value. Additionally, to combine the information of multiple miRNAs, dimension reduction methods have been applied (t-SNE, PCA and UMAP).

Claims

1. A method of monitoring the fitness level of a subject comprising the steps of:

(iii) comparing said levels determined at the different time points,

2. The method of claim 1 , wherein the level of the at least one miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20 to SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 29 to SEQ ID NO: 33 which

(iii) decreases over time indicates that the fitness level of the subject deteriorates.

3. The method of claims 1 or 2, wherein the level of the at least one miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 12 to SEQ ID NO: 19, SEQ ID NO: 23 to SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 34 which

(iii) increases over time indicates that the fitness level of the subject deteriorates.

4. A method of determining the fitness level of a subject comprising the steps of:

(ii) comparing the level of the at least one miRNA to a reference level,

wherein the at least one miRNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34. [Description (DE): wherein the reference level is the level determined by measuring at least one reference blood sample from at least one reference subject, preferably reference subject having a specific/defined fitness level, comparison allows the categorization of a subject in a specific training group, reference subject may be untrained or trained]

5. The method of claim 4, wherein the reference level is the level determined by measuring at least one reference blood sample from at least one untrained reference subject.

6. The method of claim 5, wherein

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20 to SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 29 to SEQ ID NO: 33 above the reference level indicates that the subject has a fitness level which is better than the fitness level of an untrained reference subject, or

(ii) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 12 to SEQ ID NO: 19, SEQ ID NO: 23 to SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 34 below the reference level indicates that the subject has a fitness level which is better than the fitness level of an untrained reference subject.

7. The method of any one of claims 4 to 6, wherein the reference level is the level determined by measuring at least one reference blood sample from at least one trained reference subject.

8. The method of claim 7, wherein

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20 to SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 29 to SEQ ID NO: 33 comparable with or above the reference level indicates that the subject has a fitness level which is comparable with or better than the fitness level of a trained reference subject, (ii) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20 to SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 29 to SEQ ID NO: 33 below the reference level indicates that the subject has a fitness level which is worse than the fitness level of a trained reference subject,

(iii) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 12 to SEQ ID NO: 19, SEQ ID NO: 23 to SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 34 comparable with or below the reference level indicates that the subject has a fitness level which is comparable with or better than the fitness level of a trained reference subject, or

(iv) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 12 to SEQ ID NO: 19, SEQ ID NO: 23 to SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 34 above the reference level indicates that the subject has a fitness level which is worse than the fitness level of a trained reference subject.

9. A method of determining the (predominant) training mode/type of training of a subject comprising the steps of:

(ii) comparing the level of the at least one miRNA to a reference level,

10. The method of claim 9, wherein the (predominant) training mode/type of training is endurance or strength training.

11. The method of claims 9 or 10, wherein the reference level is the level determined by measuring at least one reference blood sample from at least one reference subject who performed strength training (strength athlete).

12. The method of claim 11 , wherein

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32 above the reference level indicates that the subject makes (predominantly) endurance training, or

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO : 1 , SEQ ID NO : 9, SEQ ID NO : 11 , SEQ ID NO : 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 34 below the reference level indicates that the subject makes (predominantly) endurance training.

13. The method of any one of claims 9 to 12, wherein the reference level is the level determined by measuring at least one reference blood sample from at least one reference subject who performed endurance training (endurance athlete).

14. The method of claim 13, wherein

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32 below the reference level indicates that the subject makes (predominantly) strength training, or

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 34 above the reference level indicates that the subject makes (predominantly) strength training.

15. A method of optimizing personal training of a subject comprising the steps of:

(ii) comparing the level of the at least one miRNA to a reference level,

16. The method of claim 15, wherein the optimizing encompasses the orientation of the training in the direction of endurance or strength training.

17. The method of claims 15 or 16, wherein the reference level is the level determined by measuring at least one reference blood sample from at least one reference subject who performed strength training (strength athlete).

18. The method of claim 17, wherein

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32 above the reference level indicates that the subject (makes (predominantly) endurance training and) should optimize the training in the direction of strength training, or

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 34 below the reference level indicates that the subject (makes (predominantly) endurance training and) should optimize the training in the direction of strength training.

19. The method of any one of claims 15 to 18, wherein the reference level is the level determined by measuring at least one reference blood sample from at least one reference subject who performed endurance training (endurance athlete).

20. The method of claim 19, wherein

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32 below the reference level indicates that the subject (makes (predominantly) strength training and) should optimize the training in the direction of endurance training, or

(i) the level of the miRNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 34 above the reference level indicates that the subject (makes (predominantly) strength training and) should optimize the training in the direction of endurance training.

21. The method of any one of claims 1 to 20, wherein the blood sample is a whole blood sample.

22. The method of any one of claims 1 to 21, wherein the subject is an amateur athlete.

23. The method of any one of claims 1 to 22, wherein the subject is no professional athlete.

24. The method of any one of claims 1 to 23, wherein the level is determined by nucleic acid hybridization, nucleic acid amplification, polymerase extension, sequencing, mass spectroscopy or any combination thereof.

25. The method of any one of claims 1 to 24, wherein the level is the expression level.

26. Use of at least one polynucleotide (probe/primer, in particular primer pair) for detecting at least one miRNA in a blood sample isolated from a subject for monitoring the fitness level of the subject, wherein the at least one miRNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34 and a sequence having at least 90% sequence identity thereto.

27. Use of at least one polynucleotide (probe/primer, in particular primer pair) for detecting at least one miRNA in a blood sample isolated from a subject for determining the fitness level of the subject,

wherein the at least one miRNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 34 and a sequence having at least 90% sequence identity thereto.

28. Use of at least one polynucleotide (probe/primer, in particular primer pair) for detecting at least one miRNA in a blood sample isolated from a subject for determining the training mode/type of training of the subject,

wherein the at least one miRNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 30, and SEQ ID NO: 32 to SEQ ID NO: 34 and a sequence having at least 90% sequence identity thereto.

29. Use of at least one polynucleotide (probe/primer, in particular primer pair) for detecting at least one miRNA in a blood sample isolated from a subject for optimizing personal training of the subject,

30. The use of any one of claims 26 to 29, wherein the blood sample is a whole blood sample.

31. The use of any one of claims 26 to 30, wherein the subject is an amateur athlete.

32. The use of any one of claims 26 to 31, wherein the subject is no professional athlete.

33. A kit for monitoring the fitness level or determining the fitness level of a subject comprising:

(ii) optionally at least one reference,

34. The kit of claim 33, wherein the kit is useful for conducting the methods according to any one of claims 1 to 8, or 21 to 25.

35. The kit of claims 33 or 34, wherein the kit comprises instructions on how to carry out the methods according to any one of claims 1 to 8, or 21 to 25.

36. A kit for determining the training mode/type of training or optimizing personal training of a subject comprising:

(ii) optionally at least one reference,

37. The kit of claim 36, wherein the kit is useful for conducting the methods according to any one of claims 9 to 25.

38. The kit of claims 36 or 37, wherein the kit comprises instructions on how to carry out the methods according to any one of claims 9 to 25.

39. The kit of any one of claims 33 to 38, wherein the kit further comprises

(iii) a container, and/or

(iv) a data carrier.

40. Use of a kit for monitoring the fitness level, determining the fitness level, determining the training mode/type of training or optimizing personal training of a subject comprising:

(i) a microsampling device,

(ii) optionally an access code, and

(iii) optionally instructions on how to use the kit.

41. The use of claim 40, wherein the access code and/or the instructions on how to use the kit are comprised on a data carrier.