Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Your Manuscript ID 'sports-4067052' investigates molecular (irisin, cfDNA) and physiological adaptations in elite U18 ice hockey players. While the topic is novel and highly relevant to athlete monitoring, there are significant methodological and statistical concerns that limit the validity of the current conclusions. A major revision is necessary to address the robustness of the data and the interpretation of the findings.

A) Major Points:
1. Sample Size and Statistical Power: The longitudinal sample size (n = 12) is very small, and the cross-sectional positional analysis involves groups as small as n = 5 (defensemen). Performing inferential statistics (ANOVAs) on such small, unbalanced groups is problematic and prone to Type II errors.
In my opinion, you must explicitly acknowledge this as a major limitation. I strongly suggest framing the positional analysis as strictly descriptive/exploratory rather than inferential. Avoid strong causal claims based on this data. Consider rebranding the study as a "Pilot Study" or "Preliminary Report" in the title or abstract.

2. Control of Confounding Variables (Biomarkers): Circadian Variation: Salivary cortisol and serum markers have strong circadian rhythms. The methods section must explicitly state the exact time of day samples were collected and confirm that this was standardized across all time points (Pre vs. Early-Season). Without this, the cortisol data is uninterpretable.

3. Hemoconcentration: In longitudinal training studies, plasma volume shifts (due to hydration status or chronic training adaptations) can alter biomarker concentrations. Did the authors correct serum irisin/cfDNA for changes in plasma volume (e.g., using hematocrit/hemoglobin)? If not, this must be added as a limitation, as apparent increases might simply reflect hemoconcentration.

4. Statistical Interpretation: Your manuscript frequently discusses "trends" or differences that did not reach statistical significance (e.g., cfDNA decrease p = 0.070). While providing Effect Sizes (Cohen's d) is excellent practice, please ensure the text does not discuss non-significant results as if they were true differences. Use caution in the Discussion.
In addition, for the correlation analysis (Figure 3, n = 12), please provide Confidence Intervals (95% CI) for the correlation coefficients (r). With n = 12, an r = 0.64 has a very wide confidence interval, and the readers need to see this uncertainty.

 

B. Minor Points:
I. Dietary Control: Was food intake controlled before blood sampling? Irisin levels can be influenced by acute energy expenditure and intake.

2. Figure Quality: Please ensure all axis labels are legible and units are clearly stated in the figures.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Please note that these observations and suggestions are meant to aid in the development of the manuscript. 

The manuscript is generally well written, logically structured, and grounded in a comprehensive literature base. However, several areas would benefit from greater methodological clarity, tighter statistical framing, and improved interpretive restraint, particularly given the small sample size and exploratory nature of several analyses.

 

Study Design and Sample Size

Observations

• The longitudinal sample (n = 12) is clearly acknowledged but remains a central limitation, especially for correlational and positional analyses.
• Cross-sectional positional comparisons (e.g., forwards vs. defensemen) are underpowered, yet interpreted in relatively strong physiological terms.

Suggestions

• Emphasize more consistently throughout the manuscript that positional analyses are hypothesis-generating, not confirmatory.
• Consider explicitly labeling positional findings as exploratory trends in section headers and figure captions.
• In the Discussion and Conclusions, reduce causal language when referring to position-based differences (e.g., replace “reflects greater mechanical stress” with “may reflect”).

 

Training Load Characterization

Observations

• The training regimen is described in detail structurally (OPT phases, energy system targeting), but no objective or subjective training load metrics are provided.
• This limits interpretation of biomarker changes as adaptive versus load-driven.

Suggestions

• Explicitly acknowledge the absence of session RPE, heart-rate-derived load, GPS metrics, or volume/intensity quantification as a limitation.
• If retrospective estimates (e.g., weekly training hours, on-ice vs. off-ice distribution) are available, consider adding them descriptively to contextualize adaptations.
• In future-facing statements, recommend integrating biomarkers with load metrics rather than presenting biomarkers as stand-alone tools.

 

Biomarker Methodology and Interpretation

Observations

• The manuscript provides a thorough literature synthesis but does not sufficiently address ongoing controversies regarding irisin ELISA specificity and inter-assay variability.
• Absolute values are reported confidently despite known variability across kits.

Suggestions

• Add a short paragraph in the Methods or Limitations noting assay-related variability and the need for caution when comparing absolute concentrations across studies.
• Clarify that within-study longitudinal changes are the primary interpretive focus, rather than absolute irisin values.

Cell-Free DNA (cfDNA)

Observations

• The reduction in cfDNA is an intriguing and novel finding, but statistical significance narrowly misses the conventional threshold (p = 0.070).
• Interpretation leans strongly toward “reduced cellular stress.”

Suggestions

• Reframe cfDNA findings as moderate-effect, directionally consistent adaptations, avoiding definitive claims.
• Consider adding confidence intervals for cfDNA changes in the Results section to reinforce effect-size-based interpretation.
• Explicitly distinguish resting baseline cfDNA from acute exercise-induced cfDNA to avoid conceptual conflation.

 

Statistical Analysis and Reporting

Observations

• Both Pearson and Spearman correlations are mentioned, but justification for selecting one over the other is not always explicit.
• Multiple correlations are explored without adjustment for multiplicity.

Suggestions

• Clarify in the Methods that correlation type selection was based on normality testing at the variable level.
• Acknowledge the absence of correction for multiple comparisons and justify this decision (e.g., exploratory intent).
• Consider reporting confidence intervals for correlation coefficients, particularly in Figure 3, to emphasize uncertainty.

 

Figures and Data Visualization

Observations

• Figures are generally clear and informative, but some contain dense information that may overwhelm readers.
• Effect sizes are sometimes embedded in captions rather than integrated visually.

Suggestions

• For Figure 4, consider visually distinguishing statistically significant vs. non-significant changes more clearly (e.g., symbols or annotations).
• Ensure all figures explicitly state sample sizes in captions.
• Verify consistency in color usage and terminology across figures (e.g., “early-season” vs. “early-competition”).

 

• Standardize terminology for “early-season,” “early-competition,” and “early-training” to avoid confusion.
• Correct minor typographical issues (e.g., spacing, hyphenation, unit formatting).
• Ensure consistent reporting of VO₂max units (ml·kg⁻¹·min⁻¹).
• In the Abstract, briefly note the exploratory nature of positional analyses to manage reader expectations.

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This study analyzed physiological and molecular biological adaptations to seasonal training in elite U18 ice hockey players. The study design, which analyzed biomarkers related to maximal oxygen uptake, an aerobic performance indicator, together in human participants, is meaningful and of high value. However, there are aspects in which the interpretation of the results and the scope of application exceed the level supported by the data. Therefore, revisions and supplementation are considered necessary in order to align the presented research results with the claims of this study. The detailed review comments are as follows.

1. Abstract

In the first sentence of the abstract, the authors present rather definitively that combining performance indicators and molecular biological indicators provides benefits in monitoring adolescent team-sport athletes (“Monitoring adolescent team-sport athletes benefits from combining performance and molecular markers”). While this approach may be intuitively reasonable, the present study does not directly verify comparisons between the combined approach and single-indicator approaches, nor does it directly examine improvements in monitoring sensitivity or decision-making. Therefore, this expression should be softened to more clearly indicate that it represents a conceptual possibility rather than an empirically demonstrated fact. In addition, this sentence lacks sufficient specificity to be asserted as such a premise.

2. Study Design and Methods

This study includes both longitudinal and cross-sectional analyses and simultaneously addresses indicators with different interpretative roles, such as VO₂max, irisin, cfDNA, and cortisol. Such a complex study structure may make it difficult for readers to intuitively understand the overall flow of the study and the relationships among variables based on text alone. Adding a schematic overview (figure) that organizes the temporal structure of the study design and the interpretative position of each indicator would help convey the research intent more clearly and reduce potential misunderstandings.

3. Results and Discussion

The cross-sectional analysis by playing position has very small group sample sizes (n = 14, 5, 4), and the imbalance between groups imposes substantial limitations on result interpretation. Molecular indicators such as irisin and cfDNA are characterized by large inter-individual variability, and under such conditions, group comparisons are unlikely to achieve sufficient statistical power. Nevertheless, these limited cross-sectional findings are extended in the practical application section to examples that classify states such as overload, residual stress, and under-stimulation based on combinations of VO₂max and cfDNA, which gives the impression of exceeding the scope of the presented data.

Such state classifications are closer to a conceptual framework than to results empirically derived or validated in this study. Therefore, in the Results and Practical Applications sections, these interpretations should be clearly confined to hypothesis-generating or explanatory examples, or the level of interpretation should be substantially lowered. In particular, considering the current limited sample size and study design, expressions that directly link these classifications to actual training decision-making or applicability should be handled with greater caution.

4. Other

Throughout the manuscript, the terms “monitoring,” “assessment,” and “interpretation” are used interchangeably. Distinguishing the meanings of these terms and using them more consistently would help improve the conceptual clarity of the manuscript.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for your comprehensive and thoughtful revision of this manuscript. I am pleased to see that you have addressed most concerns raised in my previous review with considerable scientific rigor and transparency.

You have successfully addressed 7 out of 8 recommendations, implementing substantial improvements across methodology, statistical interpretation, and transparency of limitations. The manuscript now appropriately frames this work as an exploratory investigation with clearly acknowledged constraints, while maintaining its valuable contribution as the first longitudinal evidence of irisin adaptation in elite youth ice hockey players.

I also commend you for implementing several enhancements beyond my recommendations:
a) Discussion 4.1 (lines 391-393): Acknowledged ELISA specificity uncertainties and focused interpretation on within-study changes - demonstrates scientific maturity;
b) Limitations 4.3 (lines 451-454): Transparent statement about not applying multiple comparison corrections;
c) Supplementary Materials: Added Figure S1 and Figure S2;
d) Consistent unit notation improvements throughout.

On the other hand, while the scientific content is now excellent, the manuscript would benefit from professional English editing to enhance clarity and readability. Some sentences remain grammatically complex or contain minor awkward phrasing (e.g., lines 9-11, 34-35, 63-68). I recommend using MDPI's English editing service or an equivalent professional service. Please see a residual "Trend" language: Line 437-438: "forwards showed a trend toward higher irisin concentrations (928.1±310.3 vs. 683.2±68.1 ng/mL, p=0.0612)". Consider revising to: "forwards showed non-significant elevation in irisin concentrations (928.1±310.3 vs. 683.2±68.1 ng/mL, p=0.0612)". This would maintain full consistency with your exceptional revisions elsewhere.

In my opinion, this manuscript has undergone substantial improvement and now represents a scientifically sound, transparently reported exploratory investigation. You have demonstrated excellent responsiveness to peer review and scientific integrity in acknowledging limitations honestly while maintaining the novel contribution of your work. Congratulations on your rigorous revision. I believe this manuscript will make a solid contribution to the literature.

Comments on the Quality of English Language

The Manuscript ID "sports-4067052" would benefit from professional English editing to enhance clarity and readability. Some sentences remain grammatically complex or contain minor awkward phrasing (e.g., lines 9-11, 34-35, 63-68). I recommend using MDPI's English editing service or an equivalent professional service. Please see a residual "Trend" language: Line 437-438: "forwards showed a trend toward higher irisin concentrations (928.1±310.3 vs. 683.2±68.1 ng/mL, p=0.0612)". Consider revising to: "forwards showed non-significant elevation in irisin concentrations (928.1±310.3 vs. 683.2±68.1 ng/mL, p=0.0612)". This would maintain full consistency with your exceptional revisions elsewhere.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf