N6-methyladenosine (m6A) RNA modifications can be detected with high accuracy, in the form of systematic errors and decreased base-calling qualities. Specifically, we find that our algorithm, trained with m6A-modified and unmodified synthetic sequences, can predict m6A RNA modifications with ~90% accuracy. We then extend our findings to yeast data sets, finding that our method can identify m6A RNA modifications in vivo with an accuracy of 87%. Moreover, we further validate our method by showing that these ‘errors’ are typically not observed in yeast ime4-knockout strains, which lack m6A modifications. Our results open avenues to investigate the biological roles of RNA modifications in their native RNA context.The epitranscriptomics field has undergone an enormous expansion in the last few years; however, a major limitation is the lack of generic methods to map RNA modifications transcriptome-wide. Here, we show that using direct RNA sequencing,  6A RNA modifications can be detected with high accuracy using nanopore direct RNA sequencing.We currently lack generic methods to map RNA modifications across the entire transcriptome. Here, the authors demonstrate that m Systematic efforts to characterize this regulatory layer have revealed that RNA modifications are far more widespread than previously thought, can be subjected to dynamic regulation, and can profoundly impact RNA processing stability and translation10. A fundamental challenge in the field, however, is the lack of a generic approach for mapping and quantifying RNA modifications, as well as, the lack of single molecule resolution11.In the last few years, our ability to map RNA modifications transcriptome-wide has revolutionized our understanding of how these chemical entities shape cellular processes, modulate cancer risk, and govern cellular fate14; and (ii) chemical-based detection, using chemical compounds that selectively react with the modified ribonucleotide of interest, followed by reverse-transcription of the RNA fragment, which leads to accumulation of reads that have the same identical ends15. Although these methods have provided highly valuable information, they are limited by the available repertoire of commercial antibodies and the lack of selective chemical reactivities towards a particular RNA modification16, often lack single nucleotide resolution7 or require complex protocols to achieve it17, cannot provide quantitative estimates of the stoichiometry of the modification at a given site, and are often unable to identify the underlying RNA molecule that is modified.Current technologies to map the epitranscriptome rely on next-generation sequencing and, as such, they are typically blind to nucleotide modifications. Consequently, indirect methods are required to identify RNA modifications transcriptome-wide, which has been mainly approached using two different strategies: (i) antibody immunoprecipitation, which specifically recognizes the modified ribonucleotide18 and Pacific Biosciences (PacBio)3, have been proposed as a new means to detect RNA modifications in native RNA sequences. RNA modifications can be detected using PacBio in the form of kinetic changes of reverse transcriptases, which occur when the enzyme encounters a modified RNA nucleotide19. On the other hand, RNA modifications can be identified in its native RNA context using ONT, by pulling the RNA molecules through the nanopores that are embedded in a membrane. The ONT platform relies on the measurement of disruptions in the current intensity as the RNA or DNA molecule passes through the pore, which can be used to identify the transiting nucleotides. Although ONT direct RNA sequencing is already a reality21, extracting RNA modification information from ONT reads is still an unsolved challenge. RNA modifications are known to cause disruptions in the pore current that can be detected upon comparison of raw current intensities — also known as “squiggles”20. However, current efforts have not yet yielded an efficient and accurate RNA modification detection algorithm, largely due to the challenges in the alignment and re-squiggling of RNA current intensities23.To overcome these limitations, third-generation sequencing technologies, such as the platforms provided by Oxford Nanopore Technologies (ONT)N6-methyladenosine (m6A) RNA modifications in native RNA sequences, and propose a novel algorithm, EpiNano (github.com/enovoa/EpiNano), which can be used to identify m6A RNA modifications from RNA reads with an overall accuracy of ~90%. Our results provide a proof of concept for the use of base-called features to identify RNA modifications using direct RNA sequencing, and open avenues to explore additional RNA modifications in the future.As an alternative strategy, we hypothesized that the current intensity changes caused by the presence of RNA modifications may lead to increased “errors” and decreased qualities from the output of base-calling algorithms that do not model base modifications Fig. . Indeed,27. Therefore, to systematically identify the current intensity changes caused by the presence of a given RNA modification, perturbations of the current intensity signals should be measured and analyzed for each possible 5-mer (n = 1024).Previous work has shown that ONT raw current intensity signals, known as “squiggles”, can be subdivided into “events”, which correspond to consecutive 5-mer sequences shifted one nucleotide at a time 6A” instead of adenine, or unmodified ribonucleotides , while minimizing the RNA secondary structure  for each position of the k-mer   were largely nonoverlapping  using as input the base-called features from m6A-containing RRACH k-mers and unmodified RRACH k-mers (see the Methods section). We first tested whether each individual feature at position 0 (the modified site) was able to classify a given RRACH k-mer into m6A-modified or unmodified. Our results show that base quality, deletion frequency and mismatch frequency alone were able to accurately predict the modification status with reasonable accuracy   increased the accuracy of the model (88–91%) Fig. . We then6A modification status in individual sites have estimated that m6A methylation in mRNAs occurs only partially, with methylation ratios ranging from 6 to 80%28. Therefore, we wondered whether our algorithm would be able to detect m6A modifications on mixtures of methylated and unmethylated reads. To test this, we sampled reads from both m6A-modified and unmodified data sets and mixed them in different proportions, to achieve partial methylation ratios of 0 (unmodified), 5, 10, 25, 50, 75, 90, 95, and 100% (fully modified). We find that the algorithm performance is dependent on the proportion of methylated reads  of 0.72  and ime4∆ knockout strains -selected RNA from both wt and ime4∆ strains were sequenced in independent flow cells (see the Methods section), producing more than five million sequenced reads -selected RNA from EpiNano to extract base-called features for all six samples. We first analyzed the features corresponding to ~1300 known m6A-modified RRACH sites, previously identified using antibody immunoprecipitation coupled to next-generation sequencing (m6A-Seq)29. We found that base-called features at m6A-modified RRACH sites were distinct across yeast strains (wt and ime4∆), for all three metrics analyzed   and average per-read quality scores  Fig. . These rcy) Fig. . By conttes Fig. .ime4∆ strains, which also lack m6A modifications  showed intermediate mismatch frequencies between fully modified (CC 100%) and unmodified  sequences. Using linear regression, we estimated that the median stoichiometry of m6A modifications in wt strains was 12–30%  in yeast samples30. Altogether, our results reveal that nonrandom base-called “errors” present in in vivo data sets are replicable, are in agreement with in vitro results, and are correlated with the presence of m6A RNA modifications in a given site.To determine whether our trained SVM could be applied to in vivo data sets, we first investigated whether the global in vivo base-called features were consistent with those observed in vitro. We found that unmodified in vitro sequences (CC 0%) displayed similar mismatch frequencies to those observed in ons Fig. . By cont30% Fig. , which i6A-modified and unmodified in vitro constructs, to predict the transcriptome-wide m6A modification status of yeast RRACH sites, both in wt and ime4∆ data sets. A site was kept for downstream analyses if there was at least 1 read per site in each of the six samples. This criterion was met by 61,163 RRACH sites, from which 363 had been reported as “m6A-modified”, based on Illumina sequencing29. Per-site SVM predictions for each biological replicate were then merged into a single “m6A modification score” (see the Methods section). We should note that low read coverage leads to decreased accuracy  in wt and ime4∆, finding that modification scores in wt were significantly higher than those observed in ime4∆ (p = 8e–14), for the same set of sites  and unknown sites in the same strain, which do not contain m6A modifications, were relatively similar   of known m6A-modified sites  Fig. . InteresTombo22, a software originally developed for the detection of DNA modifications in nanopore sequencing data, which has recently been extended to detect RNA modifications. Identification of modifications from raw signal typically requires a two-step process: (i) re-squiggling of the raw signal to “align” all reads mapping to the same genomic location and (ii) comparison of raw current intensities across reads or samples. First, we find that the re-squiggling step used by Tombo discards ~50% of the reads . Thus, we find that, for the same set of 61,163 sites, Tombo correctly predicts known m6A sites with an accuracy of 69% and a recovery of 59%, whereas EpiNano predicts them with an accuracy of 87% and recovery of 32% . Future work will be needed to decipher whether different types of RNA modifications can be associated to distinct “error signatures”, which could be potentially used to identify the underlying RNA modification type. Thirdly, although m6A-modified RRACH k-mers globally display altered base qualities, mismatch frequencies and deletion frequencies, we should note that the contribution of each feature varies across different k-mers. For example, we find that the presence of m6A in GGACA and GGACT k-mers mainly affects the mismatch and deletion frequency, whereas in the case of GGACC, base quality and deletion frequency are the most altered features by the presence of m6A modifications , and thus lead to improved m6A RNA modification base-callers for the whole community.Here we report that RNA modifications can be identified in the form of systematic and reproducible base-calling “errors” in direct RNA sequencing data sets. These “errors” can be detected in the form of altered per-base qualities, mismatch frequencies and deletion frequencies at the modified site. We find that the method accurately detects modifications both in vitro (90% accuracy) and in vivo (87% accuracy), with an overall recovery of 32% of known sites. Despite the promising results, it is important to note, however, that the current method presents several limitations as well as ample room for improvement. Firstly, our current algorithm does not predict RNA modifications in individual RNA molecules, but rather employs information from all the reads mapping to a specific site to determine whether a given position is modified or unmodified. Secondly, our algorithm does not distinguish between different types of RNA modifications , which internally uses RNAshapes version 2.1.6 (http://bibiserv.techfak.uni-bielefeld.de/rnashapes) to predict RNA secondary structure. The final output sequence given by the software was ~10 kb long. For synthesis purposes, a total of four sequences were designed by splitting the 10 kb sequence into smaller sequences of slightly different size . Each sequence was designed with an internal strong T7 polymerase promoter, an additional BamHI site at the end of the sequence, and with all EcoRV and BamHI sites removed from the sequence   for the IVT reaction of m6A-modified RNA. IVT reaction was incubated for 4 h at 42 °C. IVT RNA was then incubated with DNAse I (Lucigen), followed by purification using the RNeasy Mini Kit (Qiagen-74104). Integrity and quality of the RNA was determined using Agilent 4200 Tapestation, to ensure that a single product band of the correct size had been produced for each IVT product  Polymerase (NEB-M0276S), following the manufacturer’s recommendations. Poly(A)-tailed RNAs were purified using RNAClean XP beads, and the addition of poly(A)-tail was confirmed using Agilent 4200 Tapestation  sequences were produced using the Ampliscribe™ T7-Flash™ Transcription Kit (Lucigen-ASF3507), using 1 µg of purified digestion product as starting material, following the manufacturer’s recommendations. ATP was replaced by ime4∆”). These strains are characterized in Agarwala et al.43. To induce synchronous meiotic entry, cells were grown for 24 h in 1% yeast extract, 2% peptone, 4% dextrose at 30 °C, diluted in BYTA  to OD600 = 0.2 and grown for another 16 h at 30 °C, 200 rpm. Cells were then washed twice with water and resuspended in SPO (0.3% potassium acetate) at OD600 = 2.0 and incubated at 30 °C at 190 rpm. Cells were isolated from SPO following 5 h and collected by 2 min centrifugation at 3000 × g. Pellets were snap frozen and stored at −80 °C for RNA extraction. Three independent biological replicates for each strain were collected.Sk1 strains used in this study were SAy841 comprising a deletion of NDT80 , and SAy966, in which both NDT80 and IME4 were deleted  dynabeads mRNA-DIRECT kit  for small mRNA amounts.Yeast total RNA samples were prepared using a modified protocol of nucleospin® 50RNA kit . Specifically, cells lysis was done in a 1.5 ml tube by adding 450 µl of lysis buffer containing 1 M sorbitol (SIGMA-ALDRICH), 100 mM EDTA and 0.45 µl lyticase (10 U/µl). The sample was incubated in 30 °C for 30 min to break the cell wall, centrifuged for 10 min at 800 × 6A-modified RNA, total of four flow cells). Each biological replicate and condition was sequenced independently in a different flow cell. For the in vivo analysis in S. cerevisiae, three biological replicates for each yeast strain (wild type and ime4∆) were sequenced, and each biological condition and replicate was sequenced in an independent flow cell .RNA library for direct RNA Sequencing (SQK-RNA001) was prepared following the ONT Direct RNA Sequencing protocol version DRS_9026_v1_revP_15Dec2016. Briefly, 800 ng of Poly(A)-tailed and capped IVT RNA — in the case of curlcakes — or 500 ng of yeast polyA + RNA were ligated to ONT RT Adapter using concentrated T4 DNA Ligase (NEB-M0202T), and was reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific-18080044). The products were purified using 1.8X Agencourt RNAClean XP beads (Fisher Scientific-NC0068576), washing with 70% freshly prepared ethanol. RNA Adapter (RMX) was ligated onto the RNA:DNA hybrid, and the mix was purified using 1X Agencourt RNAClean XP beads, washing with Wash buffer twice. The sample was then eluted in elution buffer and mixed with RNA running buffer (RRB) prior to loading onto a primed R9.4.1 flow cell, and ran on a GridION (MinION for the second replicate) sequencer with MinKNOW acquisition software version v1.14.1 (v.1.15.1 for the second replicate in the curlcake experiment). The sequencing was performed in independent days and machines, with two biological replicates for each “curlcake” experiment condition (nonmodified and m6A-modified data sets (with the largest increased in A positions) and decreased qualities . Base-called reads were filtered using NanoFilt, a component from Nanopack with settings “-q 0 -headcrop 5 -tailcrop 3”, and mapped to the 4 synthetic sequences using minimap2 with the settings -ax map-ont. Mapped reads were then converted into mpileup format using Samtools version 1.4. Read basecalling and mapping metrics can be found in Supplementary Table EpiNano (github.com/enovoa/EpiNano).To extract per-site features , BAM alignment files were converted to tab delimited format using sam2tsv from jvarkit. For each individual reference site, the mean quality of the aligned bases, the mismatch, insertion, and deletion frequency was computed using in-house scripts (available on github). To extract current intensity information from individual reads, the h5py (version 2.7.0) module in python was used to parse each individual fast5 file. Reference sequences were slided with a window size of 5 bp, and mean and standard deviation of current intensities was computed for each sliding window. All in-house python scripts used to extract the features described above are publicly available as part of 6A-modified and unmodified “curlcakes” was used as input to train a SVM. Initial training (75% of the sites) and testing (25%) of the SVM was performed with m6A-modified and unmodified curlcake reads from one replicate (rep1). Multiple kernels  were compared, and the best performing kernel was retained. The model was validated on new sequencing runs of IVT m6A-modified and unmodified sequences (rep2), which had not been used for initial training or testing of the SVM. The reported accuracy values refer to the predictions on replicate 2. The code to extract the set of features for machine learning from fastq and fast5 reads, the code for building the SVM models, as well as the trained SVM models, are publicly available in github (github.com/enovoa/EpiNano). We should note that a limitation in utilizing IVT to generate all possible 5-mers is that 5-mers that contain more than one “A” will contain more than one modification in the k-mer, e.g., AGACC will in fact be m6AGm6ACC, which are unlikely to occur in a biological context. Therefore, 5-mers that contained more than one A have been excluded from the analyses, as well as from the training set. Accuracy of the model has been computed as the sum of correct m6A modification predictions — correctly predicted m6A-modified k-mers (TP) and correctly predicted unmodified k-mers  — divided by the total number of k-mers tested.The set of extracted features of both mEpiNano was used to extract per-site features  from the mapped BAM files of the six samples sequenced . m6A-modified RRACH sites with minimum coverage of 5 reads/site were kept and scored using the previously trained SVM model. 5-mers containing more than one “A” in the motif were discarded from downstream analyses, as these k-mers had not been included in the training sets (see above). A total of 61,163 sites were analyzed for each sample and replicate, from which 363 corresponded to “known” m6A-modified sites, which had been identified using Illumina sequencing44.6A modification scores for each site were computed by merging the SVM predicted probabilities across replicates. Specifically, if the probability being modified was >0.5 in all three biological replicates , the modification score (M) was set to 1. Otherwise, the modification score was determined by computing the mean of the probabilities (pseudocode 1). Modification scores were obtained for each site, both for wild-type and ime4∆ strains.MPseudocode 1:if (s1 ≥ 0.5 and s2 ≥ 0.5 and s3 ≥ 0.5):M = 1else:M = (s1 + s2 + s3)/36A-modified” or “unmodified”, we compared the modification scores of each site, obtained for each of the two strains. Specifically, modification ratio was calculated by dividing the modification score of the wild-type strains (Mwt) and the modification score of the ime4∆ strains (Mko). A site was considered to be modified if the modification ratio was >1.5 and the modification score in wild-type strains (Mwt) was greater than 0.5 (pseudocode 2).To classify a site as “mPseudocode 2:Mwt/Mko) > 1.5 and Mwt > 0.5:if (status = modifiedelse:status = unmodified6A modification predictions — correctly predicted m6A-modified k-mers (TP) and correctly predicted unmodified k-mers (TN) — divided by the total number of k-mers tested . Positive predictive value was computed by dividing the number correctly predicted m6A-modified k-mers (TP) by the total number of m6A-modified k-mers included in the analysis (n = 363). The performance of EpiNano on yeast wt and ime4∆ samples can be found in Supplementary Table Accuracy of the predictions was computed as the sum of correct mTombo version 1.522 to align the raw signal and the base-called reads sequences (tombo resquiggle), both for wild type and ime4∆ samples. We then used the Tombo “canonical sample comparison” method (tombo model_sample_compare) to identify significant shifts in raw signals in paired data sets (wt and ime4∆) using the parameter -num-most-significant-stored 14,000,000 and -minimum-test-reads 1. M6A modification scores for each site were computed by merging the Tombo predicted probabilities across replicates. Specifically, if the probability being modified was >0.5 in all three biological replicates , the modification score (M) was set to 1, as previously done for EpiNano. Otherwise, the modification score was determined by computing the mean of the probabilities (pseudocode 1). A site was considered as modified if the modification score was >0.5. The performance of Tombo on yeast wt and ime4∆ samples can be found in Supplementary Table We first ran Further information on research design is available in the Supplementary Information NewPeer Review FileReporting SummarySource Data


Changes of direction (CODs) are key manoeuvres linked to decisive moments in sport and are also key actions associated with lower limb injuries. During sport athletes perform a diverse range of CODs, from various approach velocities and angles, thus the ability to change direction safely and quickly is of great interest. To our knowledge, a comprehensive review examining the influence of angle and velocity on change of direction (COD) biomechanics does not exist. Findings of previous research indicate the biomechanical demands of CODs are ‘angle’ and ‘velocity’ dependent and are both critical factors that affect the technical execution of directional changes, deceleration and reacceleration requirements, knee joint loading, and lower limb muscle activity. Thus, these two factors regulate the progression and regression in COD intensity. Specifically, faster and sharper CODs elevate the relative risk of injury due to the greater associative knee joint loading; however, faster and sharper directional changes are key manoeuvres for successful performance in multidirectional sport, which subsequently creates a ‘performance-injury conflict’ for practitioners and athletes. This conflict, however, may be mediated by an athlete’s physical capacity (i.e. ability to rapidly produce force and neuromuscular control). Furthermore, an ‘angle-velocity trade-off’ exists during CODs, whereby faster approaches compromise the execution of the intended COD; this is influenced by an athlete’s physical capacity. Therefore, practitioners and researchers should acknowledge and understand the implications of angle and velocity on COD biomechanics when: (1) interpreting biomechanical research; (2) coaching COD technique; (3) designing and prescribing COD training and injury reduction programs; (4) conditioning athletes to tolerate the physical demands of directional changes; (5) screening COD technique; and (6) progressing and regressing COD intensity, specifically when working with novice or previously injured athletes rehabilitating from an injury. However, GRF is a three-component vector, but a downfall of the work by Schreurs et al. [Researchers have shown that the COD angle influences the braking and propulsive force characteristics of the final foot contact (FFC) (plant phase) , 27, 29 s et al.  is only To our knowledge, only one study has directly compared the GRF characteristics of the PFC between different angled CODs , observir = 0.59) between lateral foot-plant distance and peak medial GRF during 90° cutting [Havens and Sigward  also exa cutting . ConsequThe angle of COD can also impact the velocity profile when changing direction (i.e. approach velocity and exit velocity), whereby sharper CODs result in reduced approach velocities and exit velocities , 24–26 . Havens When changing direction, the technique chosen is also angle dependent Table ; howeverInterestingly, during the redirection phase greater hip abduction, greater trunk lean angles at IC and greater hip adductor moments were observed during the 90° cut . Greaterr = 0.51, p < 0.01). However, decelerating in the rotated position would result in force absorption and loading in the transverse and frontal planes, in contrast to absorbing and decelerating the force through the sagittal plane, which may be a safer technique [Havens and Sigward  also fouechnique , 73.Although the mechanisms of ACL injury are multifactorial , COD lowSigward, Cesar and Havens  reportedSubstantiating the findings of previous studies –30, SchrAthletes unable to recognise and identify kinematic cues from their opposition or passage of play sooner  , 95, 96,It should be noted that the studies that have investigated the effect of angle on COD biomechanics Table  have allResearch from Hader et al.  has showr = 0.60–0.79, p < 0.05). Subsequently, this has large implications for the design of strength and conditioning programs and exercise selection for the enhancement of COD performance, which will be discussed in Sect. Differing from the PFC, the plant step (FFC) contains both a braking and a propulsive force component within the force-time curve , 83. CODr = 0.60–0.79, p < 0.05) between performance cutting angle and GCTs; this highlighted that sharper CODs were attributed to longer GCTs, therefore increasing braking and propulsive impulse potential.Numerous biomechanical investigations select and investigate specific angled direction changes thought to be a key mechanism of ACL injuries or pivotal movements in that sport , 111–113Collectively, based on the abovementioned findings Table , in orde−1 [−1 [−1 [−1 [−1 [−1 [−1 [−1 [−1 [−1 [Various standardised approach velocities have been administered when exploring COD biomechanics including: 3.0 m·s−1 , 51, 53,−1 [−1 , 3.6–4.4 [−1 [−1 , 37, 4.0 [−1 [−1 , 4.0–5.0 [−1 [−1 , 38, 123 [−1 [−1 , 4.5 m·s [−1 [−1 , 4.5–5.0 [−1 [−1 , 124, 4. [−1 [−1 , 126, 5. [−1 [−1 , 127 or  [−1 [−1 , 42, 56. [−1 [−1 –35 (Tabl−1) compared to slower velocities (2 and 3 m·s−1). This result is corroborated by previous studies that have reported greater KAMs between faster and slower 60° sidesteps [Vanrenterghem et al.  reportedidesteps , and 135idesteps  reportedidesteps , 92, 93 idesteps . Furtheridesteps  and rugbidesteps . Having idesteps , 58; thuidesteps , 44, 45 idesteps –84. Furt−1. Similarly, Dai et al. [−1) may not have been high enough to elicit hazardous knee joint loading connected to increased risk of ACL injury [While approach velocity is a critical factor on knee joint loading, it also directly influences the kinetic and kinematic profiles demonstrated by athletes Table . Vanrenti et al.  also demi et al.  also obsL injury , 34, 35.Changing direction is described as a multi-step action , with reCollectively, the results of the aforementioned studies highlight the role of deceleration over the PFC, and the preceding footfalls, to facilitate effective CODs. The PFC not only plays a pivotal role during sharper CODs , 60, 66,Straight line sprint speed is suggested to be a determinant of COD performance as it would be advantageous to enter and exit the COD as fast as possible , 136. Hor = −0.484, r2 = 23%) with greater approach velocities . Notably, eccentrically stronger (knee extensor peak torque) soccer players demonstrated faster COD performance (d = −2.09), greater approach velocities (d = 1.27), and greater reductions in velocity during the PFC (d = −0.94) in comparison to weaker, while greater peak and average horizontal GRFs over the PFC were also displayed by stronger athletes (d = 1.00–1.23). These findings suggest there may be an interaction between strength and speed in the facilitation of faster COD performance. Based on the results that eccentrically stronger athletes demonstrated better deceleration abilities, approached with greater velocities, and produced greater change in velocities and braking forces, the authors introduced a concept of ‘self-regulation’ regarding approach velocity (i.e. ‘a player approaches faster based on the deceleration load they know/feel they can tolerate’). Nonetheless, the ability to approach a 180° turn quickly and reduce the velocity over the PFC into the FFC is integral for faster performance, while the ability to decelerate efficiently is underpinned by eccentric strength capacity.Examining a sharper 180° COD in female soccer players, Jones et al.  inspecteThe majority of investigations have examined the influence of angle on injury risk factors during CODs , 27, 29;The technique required for faster COD performance is also angle dependent , 26, 30.In multidirectional sport it would be advantageous to perform sharp cuts from high approach velocities with minimal reductions in velocity , 24, 132Maintaining velocity and greater approach velocities are identified as determinants of COD performance , 58 and,The determinants of COD performance are multifaceted , 83, 136Table Cutting is a unilateral, multiplanar movement, and dependent on the ability to generate ML propulsive force and impulse , 30, 38,Athletes should ideally possess a solid foundation of strength (one repetition maximum back squat ≥ 1.5 × body mass) before performing complex and higher intensity plyometrics , 118, whThe biomechanical demands of CODs are ‘angle’ and ‘velocity dependent’ and are both critical factors that influence the technical execution of the COD, deceleration and reacceleration requirements, knee joint loading, and lower limb muscle activity. Thus, these two factors regulate the progression and regression in COD intensity. Specifically, faster and sharper CODs increase knee joint loading, but are also required for successful performance creating a ‘performance-injury conflict’ from a technique perspective; however, this conflict can be mediated by an athlete’s physical capacity (i.e. ability to rapidly produce force and neuromuscular control) and performing the COD with optimal mechanics. Furthermore, an ‘angle-velocity trade-off’ exists during CODs, whereby faster approaches compromise the execution of the intended COD; this is influenced by an athlete’s physical capacity. Therefore, practitioners and researchers should acknowledge and understand the implications of angle and velocity on COD biomechanics when: (1) interpreting biomechanical research; (2) coaching COD technique; (3) designing and prescribing COD training and injury reduction programs; (4) conditioning athletes to tolerate the physical demands of directional changes; (5) screening COD technique; and (6) progressing and regressing COD intensity, specifically when working with novice or previously injured athletes who are rehabilitating from an injury.


Materials for simultaneous photoelectric and thermo‐electric dual conversions and modulations, where photon can modulate the thermoelectric conversion, and temperature can modulate the photoelectric conversion, may find potential applications where light (including a laser) can remotely turn on, turn off, or modulate a thermoelectric generator, a cooler, or a temperature sensor, and vice versa, temperature (heating/cooling) can turn on, turn off, or modulate a photoelectric device such as a photo detector or a solar cell. Here, it is demonstrated that such simultaneous dual conversion or modulation can be achieved via a ternary composite, e.g., a poly‐3‐hexyl‐thiophene thin‐film doped with both phenyl‐C61‐butyric acid methyl ester and iodine. This finding may result in the development of lightweight, flexible shape, cost‐effective, renewable, environmentally friendly, biocompatible, and scalable materials, devices, and systems for clean energy harvestings  as well as light/heat dual‐sensing sensors, modulators, and controllers. Renewable, clean, cost effective, and efficient photoelectric or thermoelectric energy conversions are essential for sustainable growth of civilization, as these technologies could potentially reduce human dependence on the burning of chemical fossil fuels. Organic photoelectric charge separation can be achieved via a weak donor/acceptor pair and weakly coupled composite, also called photoelectric doping,FigureIodine doped P3HT/PCBM composite has been studied before either to improve ambient condition conductivity of the P3HT/PCBM solar cell,e is the charge of the particle, n is the mobile charge carrier density, and μ is the charge mobility. A material's thermoelectric Seebeck coefficient (S) is defined and can be experimentally measured via Equation −1) asMaterials electrical conductivity (σ) is defined in Equation n by Equation kb is the Boltzmann constant, e is the carrier charge, h is Planck's constant, m* is the effective mass of the charge carrier, and n is the majority mobile charge carrier density. In our study, carrier density n is directly related to the photoelectric or thermoelectric doping induced positive polarons on P3HT, and that the carrier mobility μ is generally and directly correlated to materials solid state packing or film morphology. If the solid state packing or film morphology are the same or similar (such as within a narrow range of temperature change), then the conductivity can reflect doping induced carrier density changes.The Seebeck coefficient is correlated to majority mobile charge carrier density Figure−2 solar simulator light, and at an elevated 39 °C temperature (ET + light) under the same light. Conductivities of undoped or pristine P3HT at these same conditions were also evaluated, and the conductivity changes are negligible , and conductivity change due to possible morphology change can also be neglected. The conductivity of the iodine doped P3HT thin films increases when heated but when illuminated, no distinguishable change is seen which is expected since there is no photo acceptor to dissociate photoinduced excitons. Figure−5 S cm−1 at room temperature in the dark to 2.54 × 10−5 S cm−1 (about 2.8% increase) at elevated temperature in the dark can be attributed mainly to the thermoelectric charge separations between iodine and P3HT. Similarly, the conductivity increase from 2.47 × 10−5 S cm−1 at room temperature in the dark to 2.63 × 10−5 S cm−1 (about 7% increase) at room temperature under light illumination can be attributed mainly to the photoelectric charge separations between P3HT and PCBM. Finally, the conductivity increase from 2.47 × 10−5 S cm−1 at room temperature in the dark to 2.67 × 10−5 S cm−1 (about 8.5% increase) at elevated temperature under light illumination can be attributed to both the thermoelectric charge separations between P3HT and iodine, and the photoelectric charge separations between P3HT and PCBM, i.e., the conductivity data confirmed that both thermoelectric and photoelectric doping processes occurred in such ternary composite.Several film samples containing pristine P3HT, P3HT/iodine (five percent mol ratio of iodine in P3HT), and an iodine/P3HT/PCBM ternary composite  was used in this demonstration due to our recent studies on doping level and/or doping ratio optimizations of such composite.FigureJ–V curves  at room temperature (25 °C) and at an elevated temperature (39 °C). Device data including open circuit voltage (Voc), short circuit current (Isc), the product of VocJsc, and power conversion efficiencies (PCEs) are listed in TableJV curves of a P3HT:PCBM binary composite film at 25 and 39 °C. Therefore, the heating incurred photo JV curve changes shown in Figure −2. The initial increase of Seebeck coefficient versus light intensity from dark to about 8 mW cm−2 could be attributed to an electron–photon coupling mechanism that are dominant at low light intensity or low charge density regime,−2 can be accounted by a dominant Seebeck versus mobile carrier density relationship expressed in Equation In order to evaluate temperature modulation of the ternary composite film photoelectric conversion, perature 9 °C. DevOur preliminary experimental data demonstrates that photon can be used to effectively modulate the thermoelectric conversions of a material, and temperature can simultaneously modulate the photoelectric conversions of the same materials if the material is doped with both a photoelectric dopant as well as a thermoelectric dopant. In this demonstration, the change of thermoelectric Seebeck coefficient between dark and highest photo modulated Seebeck is about 9.4%. With further and systematic studies and optimizations, it is feasible that light could more effectively modulate the thermoelectric conversion of the material.Our preliminary experimental results and data confirmed the feasibility of a photoelectric/thermoelectric dual conversion modulator/switching material and device. However, materials, processing, fabrications, and device parameters need to be systematically investigated and optimized in order to fit a particular application need.Potential applications of such photoelectric/thermoelectric dual conversion and dual function modulator/switching materials and devices may include, but may not limited to, an application where light (including a laser beam) can remotely turn on, turn off, or modulate a thermoelectric generator, a thermoelectric cooler, or a temperature sensor. Vice versa, the temperature (heating or cooling) can turn on, turn off, or modulate a photoelectric device such as a solar cell or a photo/radiation detector. The potential impacts of this work may result in the systematic development of lightweight, flexible shape, cost effective, renewable, environmental friendly, biocompatible, and scalable materials, devices, and systems for clean and renewable energy conversions (such as solar and waste heat energy conversions) and light/heat multiinput signal sensing and modulation.The authors declare no conflict of interest.


The results showed that dysphoric individuals reported less state anticipatory and consummatory pleasure compared with non-dysphoric individuals. Moreover, significant time-lagged associations between anticipatory pleasure and follow-up consummatory pleasure were found in the whole sample, after adjustment for current PA. The current findings thus hold considerable promise in advancing our understanding of anhedonia as well as the important role of state anticipatory pleasure in relation to depression.Anhedonia, the experience of diminished pleasure, is a core feature of major depressive disorder and is often present long before the diagnosis of depression. Most previous studies have investigated anhedonia with self-report measures of trait anhedonia or with behavioral paradigms using laboratory stimuli, and the real-time characteristics of hedonic processing in subclinical depression remain under-investigated. We used the experience sampling method to evaluate momentary experience of hedonic feelings in the context of daily life. Dysphoric ( Anhedonia, the diminished ability to experience pleasure, manifests as a transdiagnostic symptom among individuals with different psychiatric disorders . For thiHowever, accumulating evidence suggests that anhedonia is not a unitary construct. Two independent sub-components of the hedonic processing function have been revealed: anticipatory pleasure and consummatory pleasure . AnticipAlthough it is well-documented that anhedonia plays a role in depression, the role of each type of anhedonia is not well-understood. Most empirical studies on anhedonia in depression have primarily focused on consummatory pleasure. Depressed individuals have been shown to report reduced emotional reactivity to stimuli used in laboratory research , and to Compared to consummatory pleasure, less is known about anticipatory pleasure in depression. A few studies have investigated anticipatory and consummatory pleasure simultaneously, and these studies have produced mixed findings. In an MDD sample, Importantly, however, both the experimental method and self-report method have limitations in the study of anhedonia. On one hand, stimuli used in experimental studies  are often standardized to obtain more precise control of confounding factors, but because contextual information is often removed or obscured, laboratory tasks might not be analogous to the actual changing contexts of everyday life. On the other hand, in self-report studies, participants are indicated to rate their pleasure experience in response to a hypothetical situation rather than actual situation. The self-report measures of hedonic experience rely heavily on retrospective recall and therefore scores derived from these measures might be confounded by the severity of memory deficits of the clinical sample characterized by anhedonia . The redThe experience sampling method (ESM) is a technique that enables real-time and repeated sampling, which could yield more reliable and accurate estimates than single-point assessment or retrospective reports . Given tStudies have started to employ ESM to investigate the temporal features of anticipatory and consummatory pleasure in the context of daily life. The first study was conducted in schizophrenia by Although previous studies have provided preliminary evidence that patients with MDD had reduced daily experience of pleasure, it remains unknown whether these dysfunctions of hedonic processing emerge in dysphoria, who demonstrate elevated levels of depressive symptoms, but do not meet the diagnostic criteria for MDD. Dysphoric individuals showed impairment in cognitive function, such as working memory and interference control ; and theIn the affect literature, pleasure has been considered a dimension of PA. Research investigating the characteristics of PA in depression has suggested that depressive symptoms are related to lower PA both in clinical  and non-The primary goal of the current study was to understand the nature of anticipatory and consummatory pleasure in individuals with dysphoria in college-age population by using ESM. By studying individuals at risk for depression, mechanisms that underlie the future development of MDD episode may be uncovered. Furthermore, the recruitment of dysphoric individuals could overcome some of the confounding factors when studying patients with MDD, such as medication, severity of illness and episode number. MDD has its peak incidence in young adults , thus a Three hundred and forty-one college students from Wuhan, China were invited to complete the Beck Depression Inventory-II BDI-II, , which tThis study was approved by the Ethics Committee of the Central China Normal University. Written informed consent was obtained from all participants at the beginning of the baseline assessment. Participants were told they could stop participating at any time without penalty. At the end of the study participants received compensation of 30 RMB.On the first day they arrived at our laboratory, participants were administered two self-report measures. The Temporal Experience of Pleasure Scale TEPS,  is a selAn Internet link containing the set of ESM assessments was sent to each participant’s smart phone 3–4 times each day between 8:00 a.m. to 10:00 p.m., with a time interval of more than 90 min between two adjacent points, for 14 consecutive days. This resulted in a maximum of 56 events per person. Participants were asked to complete the brief assessments as soon as they received the link on their smartphone. If no response was given within 30 min or the participant spent more than 180 s to complete the assessments, the data were considered missing. Two participants from the non-dysphoric group were removed from the final analysis because their response rates were lower than 50%. Thus the final sample consisted of 49 participants in the dysphoric group and 51 participants in the non-dysphoric group. No significant group differences were observed in gender, age, or education .Momentary PA was measured with the 10 positive items  from the Positive and Negative Affect Schedule PANAS;  using thTo assess consummatory pleasure, participants firstly were asked to indicate what they were doing at that moment, by selecting from several options  and then to rate the amount of pleasure they were experiencing from 1 (“none”) to 10 (“an extremely large amount”) on a visual analog scale. To measure anticipatory pleasure at the state level, participants were asked to indicate what kind of activity they would be involved in next, and then they were asked to forecast the amount of enjoyment they would derive from that activity from 1 (“none”) to 10 (“an extremely large amount”). By including questions regarding current and future activities, the design helps to ensure that ratings for consummatory pleasure and anticipatory pleasure were based on daily events rather than memory representations. The use of brief measures is common in ESM research because excessive length can compromise response quality and reduce compliance .Group differences on demographic information and baseline self-report measures were analyzed with SPSS 23.0. The ESM data have a hierarchical structure in which within-person daily observations  are nested within between-person characteristics . Therefore, data were analyzed by means of multilevel modeling in Mplus version 7.4. Separate models were estimated for daily PA, state anticipatory pleasure, and state consummatory pleasure. Furthermore, all Level 1 predictors were person-mean centered  to separate within-person effects from between-person effects .Before running models to test our hypotheses, we first ran null models in Mplus  with PA, state anticipatory pleasure and state consummatory pleasure as the outcome variables. This type of analysis can be used to calculate intraclass correlation coefficients (ICCs), which provides an estimate of the proportion of variance in the outcome variable accounted for by the between-person level, which reflects individual differences .To examine whether dysphoric status predicted differences in anticipatory and consummatory pleasure across daily observations, we regressed the within-person parameters on dysphoric status  (see section “Model 1”).ij or anticipatory pleasure ij or consummatory pleasure ij = βj0 + rijLevel 1: PAj0 = γ00+ γ01 (dysphoric status j) + Uj0Level 2: βi represents time point, and j represents person. The outcome at Level 1 , representing the observed score of person j’s at time i, was modeled as a function of a random intercept (βj0) representing the within-person mean of the corresponding outcome variable, rij represents residual errors at within-person level. At Level-2, γ00 represents mean pleasure for the non-dysphoric group, and γ01 represents the difference in mean pleasure between the dysphoric and non-dysphoric groups. Uj0 represents residual errors at between-person level.In the equations, Next, to reveal the unique associations between momentary pleasure and dysphoria, we reran the full models, controlling for PA (see section “Model 2”).ij or anticipatory pleasure ij = βj0 + βj1 PAij + rijLevel 1: consummatory pleasure j0 = γ00+ γ01 (dysphoric status j) + Uj0Level 2: βj1 = γ10+ γ11 (dysphoric status j) + Uj1βj’s score at time i, was modeled as a function of a random intercept (βj0) representing the mean pleasure of person j, and a random slope (βj1) representing the relationship between a person j’s PA at time t and consummatory pleasure at time t.γ00 and γ10 represent the average (or fixed-effects) estimates of consummatory pleasure and PA of the non-dysphoric group, while γ01 and γ11 represent group differences of consummatory pleasure and PA between the dysphoric and non-dysphoric groups.The outcome at Level 1 , representing the observed score of person t from anticipatory pleasure at t-1 (see section “Model 3”). Additionally, consummatory pleasure at t-1 and PA at t were included as covariates separately  and simultaneously (see section “Model 6”) to control for potential confounding effects. Moreover, to examine whether dysphoria moderate these associations, a group variable indicating dysphoric status was added as the Level 2 predictor to test for group differences between the dysphoric and non-dysphoric groups  + rijLevel 1: consummatory pleasure j0 = γ00 + Uj0Level 2: βj1 = γ10 + Uj1βij = βj0 + βj1 (anticipatory pleasure t–1) + βj2 (consummatory pleasure t–1) + rijLevel 1: consummatory pleasure j0 = γ00 + Uj0Level 2: βj1 = γ10 + Uj1βj2 = γ20 + Uj2βij = βj0 + βj1 (anticipatory pleasure t–1) + βj2 (PA t) + rijLevel 1: consummatory pleasure j0 = γ00 + Uj0Level 2: βj1 = γ10 + Uj1βj2 = γ20 + Uj2βij = βj0 + βj1 (anticipatory pleasure t–1) + βj2 (consummatory pleasure t–1) + βj3 (PA t) + rijLevel 1: consummatory pleasure j0 = γ00 + Uj0Level 2: βj1 = γ10 + Uj1βj2 = γ20 + Uj2βj3 = γ30 + Uj3βij), representing a person j’s score on consummatory pleasure items at time i, was modeled as a function of a random intercept (βj0) and random slopes . βj0 represents person j’s mean score for consummatory pleasure t; βj1 represents the relationship between a person j’s anticipatory pleasure at time t-1 and consummatory pleasure at time t; βj2 represents the relationship between person j’s current consummatory pleasure and their consummatory pleasure at the prior time point in Model 4 (current PA in Model 5). βj3 represents the relationship between person j’s current consummatory pleasure and current PA. rij represents residual errors at within-person level.In the equations, the outcome at Level 1  represent the average (or fixed-effects) estimates across participants. Uj0, Uj1, Uj2, and Uj3 represent residual errors at between-person level.The Level-2 parameter estimates (γt(98) = 1.49, p = 0.14, anticipatory pleasure subscale, t(83.25) = 1.47, p = 0.15, or consummatory pleasure subscale, t(98) = 1.18, p = 0.24. Moreover, no significant group difference was observed on the SHAPS total score, t(98) = 0.65, p = 0.52. These results suggested that the dysphoric group demonstrated no attenuation of trait anticipatory or consummatory pleasure.There were no significant group differences between the dysphoric and non-dysphoric group on the TEPS total score, ij, anticipatory pleasureij, and PAij separately. Results showed that those dependent variables did not vary by age, gender, education, or time. Thus, these demographic variables and time were not included as covariates in further analyses.To determine whether age, gender, education, and time of assessments covaried with our dependent variables, we regressed these variables on consummatory pleasureThe ICCs from the null models revealed that 39.9% of the variance in PA, 34.9% of the variance in state anticipatory pleasure and 39.4% of the variance in state consummatory pleasure was at the between-person level, suggesting that both the temporal situation and dysphoric status play a role in momentary PA and the experience of pleasure in daily life. Several multilevel models were tested to examine the distinct contribution of dysphoric status to momentary pleasure respectively.p = 0.64). However, individuals in the dysphoric group reported lower levels of state anticipatory pleasure  and consummatory pleasure  than individuals in the non-dysphoric group. Moreover, group differences between the dysphoric and non-dysphoric group in state anticipatory and consummatory pleasure remained statistically significant after including PA as a covariate , but not the non-dysphoric group . In addition, the significant association between anticipatory pleasure at t-1 and consummatory at t in the dysphoric group remained unchanged after controlling for consummatory pleasure t–1 (Model 4), PA t (Model 5), or both of them (Model 6), all ps < 0.01. In the non-dysphoric group, no significant relationship was found between anticipatory pleasure and consummatory pleasure, all ps > 0.05.Results of tests of Models 3–6, namely time-lagged analyses examining associations between anticipatory pleasure and changes in consummatory pleasure in the dysphoric and non-dysphoric groups separately, are presented in ps > 0.05, see SE = 0.04, p = 0.07; after controlling for current PA, a significant association was found between anticipatory pleasure at t-1 and consummatory at t, β = 0.04, SE = 0.02, p = 0.02. In addition, the association between current PA and consummatory pleasure was also significant, p < 0.01, indicating that a higher level of anticipatory pleasure at prior time point in conjunction with higher current PA are associated with greater experience of consummatory pleasure for all participants.The moderating effects of dysphoria on these associations were not significant .Our study has several limitations. Firstly, a disproportionate number of female participants were recruited, thus our results cannot be generalized to other population. Secondly, the estimated internal consistency of anticipatory subscale of TEPS is not as strong as that of consummatory subscale in present study. Anticipatory pleasure ratings are more likely to be affected by the context and therefore show less consistency than consummatory pleasure ratings . In addiThis study makes an important contribution to the literature of anhedonia in depression by investigating the momentary level of anticipatory and consummatory pleasure during daily life activities in dysphoric and non-dysphoric individuals. The results showed that dysphoric individuals were characterized with less state anticipatory and consummatory pleasure compared with non-dysphoric individuals, irrespective of the level of daily PA. Moreover, a significant predictive relationship was found between anticipatory pleasure and consummatory pleasure in the whole sample. Our findings build on previous research highlighting hedonic deficits in depression, showing that measures of state rather than trait anticipatory and consummatory pleasure provide better estimates for hedonic processing function in dysphoria. These findings contribute to the ecological validation of the two dimension construct of anhedonia and provide specific targets that can be used to refine existing therapeutic interventions.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committee of the Central China Normal University. The patients/participants provided their written informed consent to participate in this study.XL generated the idea, designed and supervised the study, and wrote the first draft of the manuscript. Y-TZ and Z-JH helped with data collection, data analysis, and manuscript writing. X-LC and F-HY helped with data collection and data analysis. X-JS commented significantly to the draft of the manuscript. All authors contributed to and have approved the final text.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tirante Musculador®). They ingested either a capsule of placebo or caffeine (5 mg.kg-1 body mass) 24, 48 and 72 h after. Neuromuscular function and muscle power  were assessed before and after the half-squat exercise and 50 min after the placebo or caffeine ingestion at each time-point post-exercise. Sprint performance was measured at pre-test and 75 min after the placebo or caffeine ingestion at each time-point post-exercise. Maximal voluntary contraction  and twitch torque  reduced after the half-squat exercise  but returned to baseline 24 h post-exercise (P > 0.05) and were not affected by caffeine ingestion (P > 0.05). The voluntary activation  and sprint performance were not altered throughout the experiment and were not different between caffeine and placebo. However, caffeine increased height and power during the vertical countermovement-jump test at 48 and 72 h post half-squat exercise, when compared to the placebo (P < 0.05). In conclusion, caffeine improves muscle power 48 and 72 h after an eccentric-based exercise, but it has no effect on neuromuscular function and sprint performance.This study investigated the effect of caffeine on neuromuscular function, power and sprint performance during the days following an eccentric-based exercise. Using a randomly counterbalanced, crossover and double-blinded design, eleven male jumpers and sprinters (age: 18.7 ± 2.7 years) performed a half-squat exercise (4 x 12 repetitions at 70% of 1 RM), with eccentric action emphasized by using a flexible strip attached to their knees ( Strength training with eccentric emphasis is used by sprinters and jumpers to provoke in-series sarcomere hypertrophy, which could increase the velocity of muscle contraction . HoweverOnly a few studies have investigated the time course of neuromuscular fatigue after eccentric exercise –8. While+-K+-ATPase pump activity , with a Duncan post-hoc test being utilized when ANOVA detected significant main effects and/or interaction. The pre-test sprint performance before the first and second experimental blocks were compared using a paired t test in order to check if athletes had fully recovered from the previous tests and training sessions. The performance during the sprint training was then analyzed separately using a two-way, repeated-measures ANOVA Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: No**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Manuscript Number: PONE-D-19-16976Effect of Caffeine on neuromuscular function following eccentric-based exerciseGeneral CommentsAuthors carried out an interesting research regarding to the effect of caffeine on recovery of neuromuscular function, power and sprint performance following an eccentric-based exercises using a "Tirante Muscular� " in half squat exercise. Authors did a great job controlling many variables providing a broad vision when studying central and peripheral fatigue, DOMS, perceived recovery, muscle damage, CMJ and Sprint. However, the manuscript is well written and very good organized. Should be highlight why the Tirante Muscular� has been used, since it is not clear the reason that you chose this tool and not a Smith Machine or free weight. On the other hand, in the paragraph of experimental design, authors describe the sprint pre-test as “one sprint of 30 m, one of 50 m and one of 100m”. In the post- test, has not been carried out the same procedure, conversely have been performed: “1) 3 sets of 30m sprints, with a 5min rest between sets; 2) 3 sets of 50 m sprints, with a 7 min rest between sets and; 3) 3 sets of 100 m sprints, with a 10 min rest between sets”. These two protocols are different and the results cannot be compared due to the volume and the fatigue in each test. It is a limitation of this study that should be shown in the manuscript. It is possible to think that the performance in the sprint has not improved due to the fatigue that the post-test supposes. Perhaps a single series of 30m would have been enough to compare the performance in the sprint effectively. By this is important that authors explain it. Finally, only some changes I can propose to improve this manuscript. Thus, I considered that the paper could be accepted if the authors could correct mistakes pointed out. My specific comments are presented below.Specific CommentsPage 2, line 42: Before “and”, “,” should not be used. On the other hand, between “72” and “h” the “-” it is not necessary and the authors due write in the same form along the manuscript because in some case don’t use hyphen.Page 2, line 45: The use of concept baseline is not appropriated to refer to Sprint performance. This concept is well used to muscle damage, DOMS or RT, but sprint need a warm up and activation process to do the best performance. By this, change “Baseline” by “Pre-test” could be more appropriated.Page 2, line 48,52: Remove the hyphen (24 h) (48 h) (72 h).Page 3, line 73: It would be good to write a brief description of how DOMS is measured in the literature.Page 3, line 73,77,78: Remove the hyphen (24 h) (48 h) (72 h).Page 4, line 101: It is necessary write the name of Creatine Kinase before (CK) since is the first time that appear this concept during de manuscript.Page 5, line 111-112: After “years old” should be used colon not comma and it is necessary write “height:” before 180.7…. Also, it is interesting to report the Mean and SD of 1RM load or the load used corresponding with 70%1RM.Page 5, line 123: The sentence starts very similar like the sentence before. Change “study” by “research” could be a good strategy of avoid be repetitive.Page 5, line 126: Explain if the 1RM test was carried out with Tirante Muscular� .Page 6, line 133-134: Unify the way to refer days (with numbers “2” or letters “two”).Page 6, line 134: Change “baseline” by “Pre”. Also, before “and”, “,” should not be used, remove it.Page 6, line 134-135: How much time was used to recover between the sprints?Page 6, line 134-137: Was carried out some warm up before Sprints and CMJ? Could you explain it?Page 6, line 139: Explain if the half-squat exercise was performed with Tirante Muscular�.Page 6, line 157: Remove the hyphen (48 h).Page7, line 173,175: Remove the hyphen (2 min).Page 8, line 182,183: Remove the hyphens (2 min) (3 s).Page 8, line 193: Remove the hyphen (0.5 min).Page 8, line 202: Remove the hyphen (30 s).Page 9, line 209,211,212,216: Remove the hyphens.Page 10, line 240: Could you explain the stretching exercises that was carried out? It is known that passive stretching can impair the performance.Page 11, line 251: Explain if a nurse or another qualified professional took the serum extractions.Page 11, line 252: You have used “Creatine Kinase” before, the abbreviation is enough in this case.Page 11, line 258, 260: Unify the way to refer the numbers of the scale (with numbers “0” or letters “zero” and “six”). Change words by numbers.Page 12, line 284,285: Before “and”, “,” should not be used, remove it.Page 12, line 289: If you have used “a half-squat exercise” during all manuscript in this case is more appropriate than “a half-squat resistance training”. Change it.Page 13, line 299: Before “and”, “,” should not be used, remove it.Page 13, line 312: Add “exercise” after a half squat.Page 14, line 333: You can use “CMJ” instead of “countermovement jump”Page 14, line 334: If you have used “a half-squat exercise” during all manuscript in this case is more appropriate than “a half-squat resistance training”. Change it.Page 15, line 346: If you have used “a half-squat exercise” during all manuscript in this case is more appropriate than “a half-squat resistance training”. Change it.Page 16, line 368: Add “s” after “athletes’ ”Page 16, line 378: Remove the hyphens (48 h) and (72 h)Page 17, line 392: It would be interesting to complete the paragraph with a section of limitations, perhaps report about the pre and post sprints test or regarding to only use one caffeine intake protocol.Figures 3 and 4: Unify the levels of values in the Y axis of all figures. MVC has nine levels, Qtwpost has seven levels or Qtw100 has eight levels.Reviewer #2: Review to: PONE-D-19-16976; Effect of caffeine on neuromuscular function following eccentric-based exercise.Major comments.This experiment was designed to assess the neuromuscular effects of caffeine intake during the recovery phase of an eccentric-based exercise protocol that induces muscle damage. Overall, the experiment seems well designed, it contains several measurements to fulfill the objectives of the investigation and it is conveniently described in a well-written manuscript. However, the experiment has a serious flaw: it is designed to assess caffeine’s effects in the recovery phase of a muscle-damaging exercise when most, if not all, performance variables returned to basal -non-fatigued- values within 24 hours after the end of exercise. This fact is likely due to the use of a “light” eccentric exercise protocol that produced very low levels of muscle damage – a pilot study would have been helpful to detect the inefficacy of this protocol to produce moderate muscle damage”.Thus, although I found merit in the experiment and I congratulate authors for the hard work developed for this investigation, I would suggest that authors reconsider the use of “recovery” through the manuscript because this is not what they were investigating -the recovery lasted < 24 h and they did not perform any measurement in this time. For example, the conclusion of the abstract should indicate that:“Caffeine improves muscle power 48 and 72 hours after an eccentric-based exercise…”.A limitation paragraph including this aspect is highly recommended.Other comments.Introduce information about the use of caffeine in athletics. Is this common?Indicate why you selected this protocol of eccentric exercise. And discuss the lack of a relevant increase in CK .Indicate the habituation to caffeine of these individuals.The CK concentrations before exercise seems quite high . Please, discuss this in the light of previous investigations. Did they have previous muscle damage due to training?Why there is no data on jump height?Please, indicate the calculation of the sample size or at least, the statistical power with this sample of 11 individuals.A paragraph of practical applications for jumpers and sprinter might be suitable. How they should use caffeine during their training. Before all sessions? 48 h after a high-intensity session?Line 397. “sprinters” is twice**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, AttachmentREVISION.docxSubmitted filename: Click here for additional data file.AttachmentPONE REVIEW.docxSubmitted filename: Click here for additional data file. 19 Sep 2019September 16, 2019Dear Dr. Daniel BoullosaAcademic Editor of the PLOS ONEWe thank the referees for their time and effort spent in carefully reviewing our manuscript and providing such valuable comments. We also thank the editor for the opportunity to revise our manuscript: PONE-D-19-16976,“Effect of caffeine on neuromuscular function following eccentric-based exercise”. We carefully considered all referees’ suggestions and included corresponding alterations in the manuscript. Revised points in the manuscript are highlighted in yellow and a point-by-point reply is detailed below. We believe all these suggestions have improved the quality of our manuscript.Reviewer #1Authors did a great job controlling many variables providing a broad vision when studying central and peripheral fatigue, DOMS, perceived recovery, muscle damage, CMJ and Sprint. However, the manuscript is well written and very good organized. Should be highlight why the Tirante Muscular� has been used, since it is not clear the reason that you chose this tool and not a Smith Machine or free weight. On the other hand, in the paragraph of experimental design, authors describe the sprint pre-test as “one sprint of 30 m, one of 50 m and one of 100m”. In the post- test, has not been carried out the same procedure, conversely have been performed: “1) 3 sets of 30m sprints, with a 5min rest between sets; 2) 3 sets of 50 m sprints, with a 7 min rest between sets and; 3) 3 sets of 100 m sprints, with a 10 min rest between sets”. These two protocols are different and the results cannot be compared due to the volume and the fatigue in each test. It is a limitation of this study that should be shown in the manuscript. It is possible to think that the performance in the sprint has not improved due to the fatigue that the post-test supposes. Perhaps a single series of 30m would have been enough to compare the performance in the sprint effectively. By this is important that authors explain it.http://www.redalyc.org/articulo.oa?id=551656963007). We have added this information in the manuscript on Page 8, Line 179 as follow:Response: Thank you for these positive comments. We appreciate your suggestions to improve our paper. Regarding the Tirante Muscular®, we have chosen this device to increase the eccentric action during the half-squat exercise. This device is largely used by athletes in their training routine because they do not need to use excessive high external loads to increase eccentric action, avoiding injuries during eccentric-based training. A previous study showed that eccentric action is considerably increased by using this device . Participants were familiar with this accessory as they had used it in their training routine as a form to increase eccentric action without using an excessive external load, which may reduce the risk of injuries associated with eccentric-based training, mainly in a preparatory phase of an annual training periodization (17)”. Regarding the sprint training performance, we agree that pre-test cannot be directly compared to 24h, 48h and 72h recovery. We performed the pre-test sprints to check whether athletes would be fully recovered at the transition from the first experimental block (using placebo or caffeine) to the second experimental block (when supplement was inverted). Only one repetition of each distance was performed to avoid the development of fatigue and to not influence the following days tests. This procedure guaranteed that all blocks (placebo and caffeine) started with participants having fully recovered from previous tests and training and with the same recovery level in both conditions. For the post-test , more series were performed to simulate a typical sprint training session and to understand whether caffeine would be helpful to improve sprint training performance. We agree these assumptions were not clear in the paper and our statistical analysis might have not fit well to this purpose. Thus, in this new version we have compared the two pretests sprint performance just to check if there was difference in the recovery from an experimental block to another, and then separately compared the sprint performance between placebo and caffeine over the posttest period. We have included this information on Page 13, Line 294: “The pre-test sprint performance before the first and second experimental blocks were compared using a paired t test in order to check if athletes had fully recovered from the previous tests and training sessions. The performance during the sprint training was then analyzed separately using a two-way, repeated-measures ANOVA [supplement (PLA and CAF) x time ”.  Page 16, Line 362 “Pre-test sprint performance did not differ between placebo and caffeine . In addition, sprint performance for all distances  was not affected by supplement  = 0.083, P = 0.779 , ηp2 = 0.009; F = 0.203 , P = 0.663, ηp2 = 0.022 and F = 0.085, P = 0.776 , ηp2 = 0.009, Table 2). However, for both conditions, sprint performance in 50 m was faster at 72 h compared to 24 h and 48 h after exercise  = 5.395 , P = 0.014, ηp2 = 0.374) and faster in 100 m at 72 h compared to 48 h after exercise  = 6.367, P = 0.008 , ηp2 = 0.414). There was no time effect for 30 m sprint  = 3.172, P = 0.066 , ηp2 = 0.260)”.Minor: Page 2, line 42: Before “and”, “,” should not be used. On the other hand, between “72” and “h” the “-” it is not necessary and the authors due write in the same form along the manuscript because in some case don’t use hyphen.Response: Thank you for this recommendation. We have modified accordingly throughout the manuscript. Page 2, line 45: The use of concept baseline is not appropriated to refer to Sprint performance. This concept is well used to muscle damage, DOMS or RT, but sprint need a warm up and activation process to do the best performance. By this, change “Baseline” by “Pre-test” could be more appropriated.Response: We have changed “baseline” for “pre-test” when referring to sprint throughout the text, as suggested. Page 2, line 48,52: Remove the hyphen (24 h) (48 h) (72 h).Response: We have removed accordingly . Page 3, line 73: It would be good to write a brief description of how DOMS is measured in the literature.Response: This has been added on Page 12, Line 273 “The DOMS was measured with the Pain Intensity Scale ranging from 0 (no pain) to 10  (9)”. Page 3, line 73,77,78: Remove the hyphen (24 h) (48 h) (72 h).Response: We have removed accordingly .Page 4, line 101: It is necessary write the name of Creatine Kinase before (CK) since is the first time that appear this concept during de manuscript. Response: We thank the reviewer to pick up this mistake. The CK has been defined now in his first apparition “… creatine kinase (CK), a marker of muscle damage”. .Page 5, line 111-112: After “years old” should be used colon not comma and it is necessary write “height:” before 180.7…. Also, it is interesting to report the Mean and SD of 1RM load or the load used corresponding with 70%1RM.Response: We thank the reviewer to pick up these typo error. This part of the text is now reading “…”. . The 1RM load and the load corresponding to 70% of 1 RM were also inserted:“The 1RM load was 130.2 ± 18.6 kg”. .“The load at 70% of 1RM was 91.1 ± 13.9 kg”. .Page 5, line 123: The sentence starts very similar like the sentence before. Change “study” by “research” could be a good strategy of avoid be repetitive. Response: The first sentence has been changed to “(The research…)” .Page 5, line 126: Explain if the 1RM test was carried out with Tirante Muscular� .Response: We have included this information accordingly “Two preliminary sessions were designed to assess one-repetition maximal strength in a half-squat exercise (1RM) using a Tirante Musculador® ”. .Page 6, line 133-134: Unify the way to refer days (with numbers “2” or letters “two”).Response: This sentence has been changed to “After two days of low-intensity training, premeasurements were performed over the next two days”. .Page 6, line 134: Change “baseline” by “Pre”. Also, before “and”, “,” should not be used, remove it.Response: We have modified accordingly. This sentence has been changed to “premeasurements were performed…”. .Page 6, line 134-135: How much time was used to recover between the sprints?Response: We have added this information “pre-test sprints ”. .Page 6, line 134-137: Was carried out some warm up before Sprints and CMJ? Could you explain it?Response: Athletes performed a 20 min warm-up (10 minutes running + 10 minutes of dynamic stretching) before sprints. This information has been included on Page 6, Line 139 “On day 1, after a 20 min warm-up (10 minutes running + 10 minutes of dynamic stretching)”. Because athletes performed the CMJ immediately after the neuromuscular function assessment, and neuromuscular assessment was precluded by a warm-up, athletes were already warmed up and no additional warm-up was required before CMJ. Details of the warm-up before neuromuscular function assessment is provided on Page 6, Line 144 “Neuromuscular function was preceded by a warm-up (see neuromuscular function assessment section for details)”. Page 9, Line 224-227 “The neuromuscular function assessment  was preceded by a warm up composed of a 5 min jogging and 4 x 5 s isometric contractions of knee extension at ~60% of maximal subjective isometric force (30 s rest between contractions)”.Page 6, line 139: Explain if the half-squat exercise was performed with Tirante Muscular�.Response: We have included accordingly “a half-squat exercise with emphasis on eccentric action were performed using the Tirante Musculador®”. .Page 6, line 157: Remove the hyphen (48 h).Response: This has been removed accordingly .Page7, line 173,175: Remove the hyphen (2 min).Response: This has been removed accordingly .Page 8, line 182,183: Remove the hyphens (2 min) (3 s).Response: This has been removed accordingly .Page 8, line 193: Remove the hyphen (0.5 min).Response: This has been removed accordingly .Page 8, line 202: Remove the hyphen (30 s).Response: This has been removed accordingly .Page 9, line 209,211,212,216: Remove the hyphens.Response: This has been removed accordingly .Page 10, line 240: Could you explain the stretching exercises that was carried out? It is known that passive stretching can impair the performance. Response: We were aware that passive stretching impairs sprint performance; therefore, athletes performed dynamic stretching exercises, as they usually do before their training and competition. Dynamic stretching is recommended as warm up for sprint due to the similarity of the movement patterns, the consequent increase in body temperature and proprioception aid . We have added that dynamic stretching was used as warm up “The sprint training session started with a 20 min warm-up (10 minutes running + 10 minutes of dynamic stretching)”. .Page 11, line 251: Explain if a nurse or another qualified professional took the serum extractions. Response: All blood samples were collected by an accredited professional phlebotomist. We have included this information “Blood samples (8 ml) were drawn by a professional phlebotomist from the antecubital vein by venipuncture…”. .Page 11, line 252: You have used “Creatine Kinase” before, the abbreviation is enough in this case. Response: We thank the reviewer to pick up this mistake. The sentence now reads “The serum CK concentration was determined…”. .Page 11, line 258, 260: Unify the way to refer the numbers of the scale (with numbers “0” or letters “zero” and “six”). Change words by numbers.Response: This has been changed accordingly “The DOMS was measured with the Pain Intensity Scale ranging from 0 (no pain) to 10  (9). The perception of recovery was measured on the Total Quality of Recovery Scale ranging from 6 to 20, where 6 is "nothing recovered" and 20 is "fully recovered”. .Page 12, line 284,285: Before “and”, “,” should not be used, remove it.Response: We have removed accordingly .Page 12, line 289: If you have used “a half-squat exercise” during all manuscript in this case is more appropriate than “a half-squat resistance training”. Change it. Response: The sentence now reads “Creatine kinase, delayed onset muscle soreness and perceived recovery at baseline, and at 24, 48 and 72 h later a half-squat exercise” . .Page 13, line 299: Before “and”, “,” should not be used, remove it.Response: We have removed accordingly .Page 13, line 312: Add “exercise” after a half squat.Response: We have added accordingly. The text now reads “Neuromuscular function assessed before and after a half-squat exercise”. .Page 14, line 333: You can use “CMJ” instead of “countermovement jump”Response: The sentence now reads “Countermovement jump test performed before and after a half-squat exercise”. .Page 14, line 334: If you have used “a half-squat exercise” during all manuscript in this case is more appropriate than “a half-squat resistance training”. Change it. Response: We have changed accordingly. This text now reads “. Countermovement jump test performed before and after a half-squat exercise”. .Page 15, line 346: If you have used “a half-squat exercise” during all manuscript in this case is more appropriate than “a half-squat resistance training”. Change it. Response: We have changed accordingly. This text now reads. “Sprint performance at 24, 48 and 72 h later a half-squat exercise”. .Page 16, line 368: Add “s” after “athletes’ ”Response: We have modified accordingly .Page 16, line 378: Remove the hyphens (48 h) and (72 h)Response: This has been removed .Page 17, line 392: It would be interesting to complete the paragraph with a section of limitations, perhaps report about the pre and post sprints test or regarding to only use one caffeine intake protocol. Response: A new paragraph has been added to address limitations. We have added the low level of pain generated by our resistance training protocol (suggestion of reviewer 2) and using only one caffeine intake protocol (your suggestion) as limitations. However, based in your first comment, we have reanalyzed the sprint data and felt we have solved the problem of the differences in the numbers of sprints between pre and post sprints . Thus, we think this is now not properly a limitation. The limitation paragraph reads  “There are some limitations in the present study that should be mentioned. Our resistance training protocol generated mild to moderate muscle pain, resulting in no impairment of subsequent training capacity. Thus, whether caffeine would be useful when greater muscle pain is present deserves further investigation. Another potential limitation is that experimental conditions were performed only once each (one for placebo and another one to caffeine). Repetitions of the experimental blocks may have provided additional information regarding the reproducibility of our findings”.Figures 3 and 4: Unify the levels of values in the Y axis of all figures. MVC has nine levels, Qtwpost has seven levels or Qtw100 has eight levels.Response: We have modified Y- axis in figures 3 and 4 and now all Y-axis have nine levels.Reviewer #2:This experiment was designed to assess the neuromuscular effects of caffeine intake during the recovery phase of an eccentric-based exercise protocol that induces muscle damage. Overall, the experiment seems well designed, it contains several measurements to fulfill the objectives of the investigation and it is conveniently described in a well-written manuscript. However, the experiment has a serious flaw: it is designed to assess caffeine’s effects in the recovery phase of a muscle-damaging exercise when most, if not all, performance variables returned to basal -non-fatigued- values within 24 hours after the end of exercise. This fact is likely due to the use of a “light” eccentric exercise protocol that produced very low levels of muscle damage – a pilot study would have been helpful to detect the inefficacy of this protocol to produce moderate muscle damage”. Thus, although I found merit in the experiment and I congratulate authors for the hard work developed for this investigation, I would suggest that authors reconsider the use of “recovery” through the manuscript because this is not what they were investigating -the recovery lasted < 24 h and they did not perform any measurement in this time. For example, the conclusion of the abstract should indicate that: “Caffeine improves muscle power 48 and 72 hours after an eccentric-based exercise…”. A limitation paragraph including this aspect is highly recommended.Response: We are happy that you have appreciated our experiment design and the quality of writing of the paper. We also thank you for your suggestions to improve our paper. We agree that our eccentric exercise protocol was not intense enough to provoke long impairment in our main outcomes. A pilot study would be helpful, but as we recruited athletes involved in national and international competitions and they were engaged in experiments taking 3 weeks, we were afraid to propose a longer experimental design and impair their preparation and carry to decline participation. In addition, we were afraid to propose a more intense protocol to produce larger muscle damage because they were in the preparatory phase of an annual training periodization; therefore, more susceptive to muscle injuries if using intense muscle damage protocol. To not miss the opportunity to test performance in athletes of this level, we ascertain this light protocol with their coach. However, we recognize this is a limitation and the term “recovery” should not be used. Based on your suggestion, we have removed the use of recovery throughout the manuscript and replaced to “after a half-squat exercise with eccentric emphasis” or something similar. Conclusion in the abstract was also altered as suggested  to “In conclusion, caffeine improves muscle power 48 and 72 h after an eccentric-based exercise, but it has no effect on neuromuscular function and sprint performance”. We have also added a paragraph in the discussion recognized this point as a limitation, as suggested  “There are some limitations in the present study that should be mentioned. Our resistance training protocol generated mild to moderate muscle pain, resulting in no impairment of subsequent training capacity. Thus, whether caffeine would be useful when greater muscle pain is present deserves further investigation. Another potential limitation is that experimental conditions were performed only once each (one for placebo and another one to caffeine). Repetitions of the experimental blocks may have provided additional information regarding the reproducibility of our findings”.Based on the data presented, I have the following comments/questions.1. Introduce information about the use of caffeine in athletics. Is this common?Response: Thank you for this question. Yes, it was. Since caffeine has been removed from the banned list of substances of World Anti-Doping Agency (WADA), there are a progressive increase in caffeine use as an ergogenic resource by athletes of various sports, including athletics . We have included a sentence in the introduction providing this information  “Following its removal from the World Anti-Doping Agency (WADA) Prohibited List in 2004, the use of caffeine for track and field athletes increased substantially (12). The use of caffeine has been reported to be large in national/international level athletes (~48%) and in power athletes (~57%) (12)”. 2. Indicate why you selected this protocol of eccentric exercise. And discuss the lack of a relevant increase in CK .Response: We have included an explanation about this choice on Page 8, Line 200-204 “We ascertain this protocol with athetes’s coach to simulate their training routine. We chose a protocol able to provoke moderate rather than a more intense DOMS because athletes were in a preparatory phase of an annual training periodization; therefore, protocol to induced more intense DOMS would make athletes more susceptive to muscle injuries”. We have also included in the discussion section the lack of a relevant increase in CK and the comparison with values after a marathon, as suggested  “We also found an increase in CK levels 48 h after exercise, regardless of the supplement ingested. This finding is in agreement with a study showing that CK peaks within 48 h after a marathon (24). However, our CK level 48 h after the half-squat exercise was much lower than those reported 24 h after a marathon (434-844 U/L) (24). This is expected because CK levels after prolonged exercise such as marathon can reach up to 50 times the rest values due to the greater muscle damage caused by this kind of activity (24)”.3. Indicate the habituation to caffeine of these individuals.Response: We have included the athletes’ daily caffeine consumption on Page 5, Line 117 “low-to-moderate habitual caffeine consumption (< 80 mg.day-1) (16)”. 4. The CK concentrations before exercise seems quite high . Please, discuss this in the light of previous investigations. Did they have previous muscle damage due to training?Response: Thank you for this question. The baseline CK upper limit for general population is 174 U/L. We found an average baseline value of 330 U/L, which suggests a slightly already elevated CK. Unfortunately, daily training results in persistent CK elevation in athletes, which makes hard to find a “true” baseline. We tried to control this by providing two days of low-intensity training before blood sample, as it would be difficult to maintain high performance athletes without any kind of training. Although we have not got values below 174 U/L, the values were quite close (~ 330 U/L) and not different between supplements, which suggests that any effect of previous training on CK as similar across experimental conditions. We have added a discussion about this on Page 17, Line 397-406 “The CK after an eccentric exercise peaks 1–4 days and can remain elevated for several days . An important issue is that daily training results in persistent CK elevation in athletes, with resting values being higher than in non-athletes (25). In the present study, even providing two days of low-intensity training before taking blood sample, we found baseline CK values ranging from 199 to 514 U/L, which are slightly above the upper reference limit established to general population (174 U/L) (26). Because it is not possible to maintain athletes without any kind of training for days, a “true” CK baseline value is hard to find in athletes. However, the values found in the present study are in agreement with reference values proposed for male athletes (82-1083 U/L) (25)”.5. Why there is no data on jump height?Response: We have included data from jump height in this new version, as suggested. We have changed information in Methods section  “The CMJ height was calculated from flying time, while CMJ power calculated from flying time and body mass, using a commercial software .” and Results section  “There was a significant supplement vs. time interaction for CMJ performance. The height  = 3.299, P = 0.024, ηp2 = 0.32, Fig 4A) and power  = 2.903, P = 0.039, ηp2 = 0.29, Fig 4B) during the CMJ were higher 24 h after exercise compared to baseline and post-exercise in both supplements . However, height and power during the CMJ 48 and 72 h after exercise remained above baseline values only when caffeine was ingested (P = 0.001). The height and power during the CMJ 48 and 72 h after exercise was also higher in caffeine compared to the placebo (P = 0.010)”. We have also included CMJ height in figure 4.6. Please, indicate the calculation of the sample size or at least, the statistical power with this sample of 11 individuals.Response: We have added a paragraph in the statistical analysis section explaining the calculation of the sample size “Because of the lack of data regarding the caffeine effects on neuromuscular function post an eccentric-based exercise in athletes, the required sample size was estimated using an effect size (ES = 0.37) reported in a meta-analysis investigating the effect of caffeine ingestion on muscular strength (23). With an alpha of 0.05 and a desired power of 0.80, the total sample size necessary to achieve statistical significance was estimated to be 10 participants. However, the starting sample size was increased to 11 participants, assuming that 10% might drop out during the data collection. The sample size calculation was performed using G*Power software . .7. A paragraph of practical applications for jumpers and sprinter might be suitable. How they should use caffeine during their training. Before all sessions? 48 h after a high-intensity session?Response: Thank you four this suggestion. A new paragraph has been added to address practical applications  “Our results has considerable practical relevance as they are indicating that caffeine can optimize jumping performance in well-trained athletes even when a certain degree of muscle damage and DOMS are present. Thus, ingestion of anhydrous caffeine in some jump training sessions may be an interesting strategy to improve training quality and performance of these athletes”.8. Line 397. “sprinters” is twice.Response: This typo error has been fixed accordingly. The text now reads “(sprinters and jumpers)”. .AttachmentResponse_to_Reviewers.pdfSubmitted filename: Click here for additional data file. 23 Oct 2019Effect of caffeine on neuromuscular function following eccentric-based exercisePONE-D-19-16976R1Dear Dr. Lima-Silva,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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