Metabolic, Cardiovascular, and Stress Biomarker Adaptations to Breath-Hold Training in a National-Level Swimmer: A Signal-Generating Single-Case Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Well designed and executed single case study with interesting integration of 8 weeks BHT in elite female swimmer. Carefully planned study that should lead to larger scale cohort study to investigate individual tailoring of BHT into regular swimming regime. Clear acute physiological adaptations (lactate, HR, VO2max, DLCO, reduced salivary stress hormones, HRV, etc. 

Specific comments

Static apnea should include several preparatory apneas in addition to maximal inspiratory breath holds, that prolong easy going phase and struggle phases. Has your athlete ever reached start of the involuntary breathing maneuvers at the end of easy going phase?

Cycle ergometry should be replaced by in-water testing including VO2max test to test sport specific adaptations.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General comments

Congratulations on the work conducted. In my opinion, this is a very interesting study that provides valuable insights and, importantly, may serve as a strong foundation for future research. Given that this is a case study, I believe it is essential to include the full training protocol, either within the main manuscript or as supplementary material, to ensure transparency and reproducibility. Additionally, the Introduction and Discussion sections would benefit from being divided into shorter paragraphs to improve readability. These points, along with several other specific comments, are detailed below.

Specific comments:

Line 28: DLCO has not been introduced.

Introduction: The authors should explain what breath-hold training consists of. This description may fit well around line 43.

Line 43: Please include a reference to support this statement.

Lines 41–69: The introduction is well written; however, it should be structured into multiple paragraphs. I recommend splitting this large paragraph into smaller sections to improve readability.

Lines 69–78: This information belongs in the Methods section rather than the Introduction.

Line 78: The authors mention aspects in the aim that have not been previously introduced. It is important that the reader understands the rationale behind measuring these variables. This does not require an explicit explanation but rather sufficient background context.

Line 89: The acronyms DLCO and CPET have not been previously defined.

Lines 95–99: Given that the participant is a swimmer, I recommend using a standardized classification model to characterize athletic level in swimming. See Ruiz-Navarro et al.:

Ruiz-Navarro, J. J., López-Belmonte, Ó., Gay, A., Cuenca-Fernández, F., & Arellano, R. (2023). A new model of performance classification to standardize research results in swimming. European Journal of Sport Science, 23(4), 478–488.

BHT intervention: This section should be described in greater detail. From a coaching perspective, it is important to clearly understand the procedures in order to replicate them.

Lines 154–155: Please clarify what is meant by “at least two reproducible maneuvers.”

Cardiopulmonary exercise testing: What type of exercise was used? Was it an incremental cycling test? This should be clearly specified.

Lines 162–163: Peak oxygen uptake (VO₂peak) and VO₂max are not equivalent. Achieving VO₂max requires specific criteria, including the presence of a plateau. Please clarify whether VO₂max was truly reached or if you are referring to VO₂peak.

Lines 173–174: How was this determined? Was it directly measured, or was it assumed that hormonal levels were similar during that period?

Line 181: For lactate concentration, the standard abbreviation is [La⁻]. Please use this notation.

Lines 197–198: Based on the reported times, this swimmer would likely be classified as Level 3 (national level). There is no need to overemphasize the competitive level.

Line 219: Heart rate is a discrete variable, not continuous. Fractional values (e.g., half beats) are not physiologically meaningful.

Results: This section should present the data only. Interpretation, while valuable, should be reserved for the Discussion.

Discussion: Please structure this section into multiple paragraphs to improve readability.

Line 286: This may not be a favorable outcome for sprinters, who typically aim to develop anaerobic energy systems to maximize rapid energy production.

Line 264: The authors should avoid overly definitive conclusions. A key limitation is present: how can it be determined that the observed changes are due to apnea training rather than the overall training program?

Line 314: Please include a reference to support this statement. This may not apply to sprinters in 50 m events, for example. However, sprinters often train apnea, as breathing is minimized to enhance performance. As shown by McCabe et al. and Pedersen and Kjendlie, each breath can impact performance by approximately 0.02 s:

• Pedersen T, Kjendlie L. The effect of breathing action on velocity in front crawl sprinting. Portuguese Journal of Sport Science. 2006;6:75–77.
• McCabe CB, Sanders RH, Psycharakis SG. Upper limb kinematic differences between breathing and non-breathing conditions in front crawl sprint swimming. Journal of Biomechanics. 2015;48:3995–4001.

Line 371: This is an important point. Why was no swimming-specific performance test included? For example, a 50 m maximal effort test would have been highly relevant.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,
Thank you for the opportunity to review your manuscript entitled “Metabolic, Cardiovascular, and Stress Biomarker Adaptations to Breath-Hold Training in an Elite Swimmer: A Signal-Generating Single-Case Study”. The manuscript presents an eight-week individualized breath-hold training intervention integrated into the regular training routine of an elite female freestyle swimmer. The study examines pre- to post-intervention changes across an integrated set of physiological and biochemical outcomes, including lactate and heart rate responses, salivary cortisol and alpha-amylase, pulmonary function, diffusing capacity, and cardiopulmonary exercise performance.
The topic is appropriate for the journal and the signal-generating orientation is potentially valuable. However, several aspects of the manuscript require revision before the findings can be considered fully balanced and adequately supported.
Please find below my comments aimed at helping you strengthen the manuscript. 
General comments
Strengths: The ecological nature of the intervention is a strength, as the breath-hold training program was embedded within the athlete’s real training environment. The combination of dry static apnea, in-water dynamic apnea, lactate testing, heart rate responses, pulmonary function, CPET variables, and salivary biomarkers provides a broad monitoring framework. The inclusion of salivary cortisol and alpha-amylase is particularly valuable because neuroendocrine responses to breath-hold training remain less commonly studied in applied sport settings.
Limitations: The principal limitation is the single-case design without a control condition, repeated baseline, or direct swimming performance outcome after the intervention. Therefore, the manuscript should avoid language implying efficacy, causality, or generalizability. Several mechanistic interpretations are physiologically plausible, particularly those related to changes in lactate handling, cardiovascular and autonomic regulation, pulmonary gas exchange, oxygen delivery and utilization, and neuroendocrine stress reactivity. However, these mechanisms were not directly assessed and should therefore be presented as hypotheses rather than demonstrated adaptations. 
Major comments
The manuscript is appropriately framed as a signal-generating single-case study, and this orientation is suitable for the nature of the work. However, this framing should be maintained more consistently throughout the manuscript. The observed pre- to post-intervention changes are interesting and potentially meaningful, but they cannot establish that breath-hold training independently caused the reported improvements. Because the athlete continued regular elite training, other factors such as normal training progression, test familiarization, recovery status, menstrual cycle effects, nutrition, sleep, tapering, and day-to-day biological variability cannot be excluded. For this reason, statements suggesting that BHT enhanced performance capacity, stress resilience, autonomic control, pulmonary gas exchange, or metabolic efficiency should be moderated and presented as plausible interpretations rather than demonstrated effects.
The intervention itself is relevant and feasible, but the methodological description should be strengthened to improve reproducibility and interpretation. Please, clarify whether BHT was added to the athlete’s regular training load or substituted for part of it, since changes in total training load may have contributed to the observed responses. The weekly progression of the protocol should also be described more clearly, including apnea duration, recovery intervals, breathing pattern progression, swimming intensity, perceived exertion, heart rate responses, and any objective safety monitoring. Although the manuscript states that international safety guidelines were followed, the specific safety procedures used during in-water apnea should be reported with greater precision.
The statistical approach is a central issue that requires revision. The use of Wilcoxon signed-rank tests, Cohen’s d, and statements about large or very large effect sizes is problematic in a single-participant pre-post design. Repeated workloads, time points, duplicate assays, or beat-to-beat values should not be treated as independent observations. The analysis would be more appropriate if presented primarily as descriptive and hypothesis-generating, focusing on raw values, percent changes, visual inspection, technical error, coefficients of variation, and whether the observed changes exceed expected measurement variability. Similarly, wording such as “significant reductions” in the abstract and results should be avoided unless the statistical unit and assumptions are clearly justified. I suggest clarifying what the reported standard deviations represent, since heart rate SD appears to reflect variability within the final 30 seconds of a stage or across stages, whereas salivary biomarker SD reflects duplicate assay variability. These are different sources of variation and should not be presented as equivalent biological variability.
The reduction in lactate concentration at matched workloads and the increase in W4 are among the most interesting findings of the manuscript. Nevertheless, the interpretation should remain cautious. These changes should not be attributed to improved lactate clearance, mitochondrial efficiency, MCT-mediated transport, or enhanced lactate oxidation because these mechanisms were not directly measured. Although some sections already acknowledge this limitation, the same caution should be applied consistently across the abstract, results, discussion, and conclusion. The most appropriate interpretation is that the athlete showed reduced lactate accumulation during the post-intervention test, while the underlying mechanism remains unresolved.
The pulmonary and CPET findings also require a more conservative interpretation. The reported increases in DLCO, VO2max, and oxygen pulse may be meaningful, but the authors should discuss whether these changes exceed expected test-retest variability. Oxygen pulse is an indirect index and cannot distinguish changes in stroke volume from changes in peripheral oxygen extraction without direct cardiovascular or arteriovenous oxygen difference measurements. Similarly, DLCO alone cannot determine whether changes reflect membrane diffusion, capillary recruitment, or normal measurement variation. These outcomes should therefore be described as supportive physiological observations rather than confirmed mechanisms.
The inclusion of salivary cortisol and alpha-amylase is a valuable aspect of the study, but the neuroendocrine interpretation should also be moderated. The observed reduction in salivary biomarker responses may suggest attenuated neuroendocrine reactivity to the post-intervention exercise test. However, claims of HPA-axis or SAM-system recalibration should be softened unless supported by additional evidence. You should also clarify the saliva sampling time points, the AUC calculation method, intra-assay and inter-assay variability, and whether salivary flow rate was measured or controlled.
Finally, the Discussion is scientifically interesting but longer than necessary for a single-case report. Several mechanisms are discussed in a way that may exceed the evidence generated by the study. The discussion would be stronger if it clearly separated the observed findings from plausible mechanistic explanations and practical implications. Table 5 is useful as a proposed monitoring framework, but it should be explicitly presented as a practical suggestion derived from the literature and the present case, not as a validated outcome of this study. In addition, the reference list requires careful manual verification, as several entries appear to contain incorrect bibliographic information, including year, journal, volume, or page range. The references related to apnea as a training method, apnea effects on swimming coordination, voluntary hypoventilation in swimmers, and myoglobin oxygen desaturation should be checked carefully, and each citation should be verified against the original source to ensure that it supports the specific claim for which it is used.
Minor comments 
    Please revise the author list formatting. In lines 7-8, there are punctuation and spacing inconsistencies, including a double comma after “Paride Vasco 2,,” and missing spacing in author affiliations.
    Please correct the age inconsistency. The abstract describes the athlete as a 23-year-old female swimmer in line 23, and the Methods section repeats this in lines 93-94. However, Table 1 reports the athlete’s age as 22 years. 
    Please revise the sentence in the abstract reporting the W4 result. In lines 28-30, the phrase “a 20 W increase in power at 4 mmol·L−1. Lac” contains an awkward punctuation break and should be corrected for clarity.
    Please standardize the terminology used for lactate. The manuscript uses “Lac” in lines 26 and 79, “lactate kinetics” in lines 88-89, and “blood lac” in line 211. A consistent term such as “blood lactate” should be used throughout.
    Please verify the reference ranges and units in Table 1. In particular, the TSH unit is reported as ng·mL−1, which should be checked because TSH is commonly reported as mIU/L or µIU/mL. The reference ranges for body composition variables and phase angle should also be supported by appropriate references.
    Please revise Table 3. The table note is duplicated, and the presentation of lactate and W4 as mean ± SD should be reconsidered if these values were derived from single test observations or interpolation. In addition, the HR SD values differ between Table 3 and the text: Table 3 reports 129.7 ± 6.2 and 124.9 ± 5.4 bpm, whereas lines 218–219 report 129.7 ± 5.8 and 124.9 ± 5.1 bpm.
    Please avoid using “patient” in the informed consent statement. In lines 426-427, “participant” or “athlete” would be more appropriate for this case study.
    Please use one spelling style consistently. The manuscript includes “standardized” in lines 84 and 165, but also “optimisation” in line 415. 
    Please clarify the acknowledgment statement in lines 431-432. The sentence indicating that the authors “reviewed and edited the output” should specify the nature of the output or tool used.
I truly hope these comments help further strengthen the manuscript.

Comments on the Quality of English Language

The manuscript is readable overall. A careful language revision would help the manuscript communicate its findings in a more balanced and polished manner.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Congratulations on the work done. In my opinion the manuscript now reads better and provides greater insights for the reader.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,
Thank you for the opportunity to second-round review your manuscript entitled “Metabolic, Cardiovascular, and Stress Biomarker Adaptations to Breath-Hold Training in an Elite Swimmer: A Signal-Generating Single-Case Study”. The manuscript presents an eight-week individualized breath-hold training intervention in an elite female freestyle swimmer and examines pre- to post-intervention changes in lactate and heart rate responses, salivary stress biomarkers, pulmonary function, diffusing capacity, and cardiopulmonary exercise testing outcomes.
The revised version shows improvement compared with the previous submission. In particular, the breath-hold training protocol is now described with greater detail, the CPET procedure is clearer, and the salivary biomarker sampling strategy is more transparent. These revisions improve methodological reproducibility and make the manuscript easier to follow. Nevertheless, several central concerns raised in the previous review remain only partially addressed. The manuscript is still limited by a single-case pre-post design, and some statistical and mechanistic interpretations remain stronger than the evidence can support.
Please find below my general and section-specific comments aimed at strengthening your work. 
General Comments
Strengths: The revised manuscript is more complete methodologically than the previous version. The authors have added useful detail regarding preparatory apneas, the struggle phase, involuntary breathing maneuvers, recovery intervals, progression rules, and dynamic apnea training. The CPET section has also improved by providing a clearer protocol and by using VO2peak rather than VO2max, which is more appropriate for this testing context. The salivary biomarker section now includes more precise information about sampling time, recovery timing, menstrual-cycle standardization, and pre-analytical control. These changes strengthen the practical and methodological value of the manuscript.
Weaknesses: Despite these improvements, the revision does not fully resolve the main interpretive and analytical concerns. The manuscript still presents some outcomes in a way that implies efficacy, causality, or confirmed physiological adaptation, even though the design does not include a control condition, repeated baseline, direct swimming performance assessment, or direct mechanistic measurements. The statistical analysis also remains problematic for a single-participant case study, particularly because Wilcoxon testing, Cohen’s d, and significance-oriented wording are still retained. In addition, several reference and editorial issues remain unresolved.
Major Comments
The revised Methods section is clearly stronger than before. The additional description of static apnea preparation, the struggle phase, involuntary breathing maneuvers, underwater distance progression, breathing-frequency progression, recovery intervals, and supplementary protocol material improves reproducibility. However, one important issue remains insufficiently clarified. The manuscript should explicitly state whether the BHT sessions were added to the athlete’s usual training load or substituted for part of the regular training program. This distinction is essential because changes in total training load could have contributed to the observed physiological responses.
The safety description for in-water apnea also requires further refinement. The manuscript states that sessions were supervised by certified coaches and conducted according to safety guidelines, but the practical safety procedures remain general. Please specify whether pre-apnea hyperventilation was prohibited, whether one-to-one supervision was used during in-water apnea, whether rescue procedures were available, and whether any objective safety monitoring, such as SpO2was performed. This information is important because the intervention involves repeated breath-hold exposures in an aquatic environment.
The statistical approach remains the most important unresolved issue. The revised manuscript still includes Wilcoxon signed-rank tests, Cohen’s d, and statements about effect sizes, despite the single-participant pre-post design. Repeated workloads, time points, duplicate assays, or beat-to-beat values should not be treated as independent observations. The analysis should be reframed as descriptive and hypothesis-generating, emphasizing raw values, percent changes, visual inspection, technical or analytical variability, coefficients of variation, and whether changes plausibly exceed expected measurement error. Phrases such as “significant reductions” and “large effect sizes” should be removed or substantially qualified.
The manuscript still contains causal and mechanistic wording that is too strong for the design. The observed reductions in lactate and heart rate, together with changes in VO2peak, oxygen pulse, DLCO, salivary cortisol, and salivary alpha-amylase, are interesting and may generate useful hypotheses. However, they do not demonstrate that BHT independently enhanced metabolic efficiency, autonomic control, pulmonary gas exchange, stroke volume, peripheral oxygen extraction, or stress resilience. These interpretations should be consistently presented as plausible physiological explanations rather than confirmed adaptations.
The interpretation of lactate responses should remain more cautious. The reduction in lactate concentration at matched workloads and the increase in W4 are among the most relevant findings of the manuscript. However, the underlying mechanism remains unresolved because lactate turnover, lactate transport, mitochondrial function, and lactate oxidation were not directly assessed. The most appropriate interpretation is that the athlete showed reduced lactate accumulation during the post-intervention test. Claims related to lactate clearance, mitochondrial efficiency, MCT-mediated transport, or enhanced oxidation should be clearly framed as hypotheses.
The pulmonary and CPET findings also need a more conservative interpretation. The revised use of VO2peak is appreciated, but the reported changes in DLCO, VO2peak, and oxygen pulse should be discussed in relation to expected test-retest variability. Oxygen pulse remains an indirect index and cannot distinguish stroke volume from peripheral oxygen extraction without direct cardiovascular or arteriovenous oxygen difference measurements. Similarly, DLCO alone cannot determine whether the observed change reflects membrane diffusion, capillary recruitment, or normal measurement variability.
The salivary biomarker methods have improved, but several analytical details remain necessary. Please report the AUC calculation method, intra-assay and inter-assay coefficients of variation, and whether salivary flow rate was measured or controlled. This is particularly relevant for salivary alpha-amylase, which can be influenced by flow rate and collection conditions. The reduction in salivary cortisol and alpha-amylase may suggest attenuated neuroendocrine reactivity to the post-intervention exercise test, but claims of HPA-axis or SAM-system recalibration should be softened unless supported by additional evidence.
The revised limitations section is helpful, but the caution expressed there should be reflected more consistently across the Abstract, Results, Discussion, and Conclusions. At present, the manuscript acknowledges important limitations near the end, but earlier sections still use stronger wording than the study design supports. The overall tone should be harmonized so that the manuscript clearly presents itself as a signal-generating case study rather than evidence of intervention efficacy.
The reference list still requires careful verification. Some entries appear to retain incorrect bibliographic information involving journal, year, volume, or page range. The references related to apnea as a training method, apnea effects on swimming coordination, voluntary hypoventilation in swimmers, and myoglobin oxygen desaturation should be checked against the original sources. Each citation should also be verified to ensure that it supports the specific statement for which it is used. 
Minor Comments
    The title still contains inconsistent capitalization, particularly the use of “And” in the middle of the title. Please revise according to the journal’s style.
    The author list still contains formatting issues, including the double comma after “Paride Vasco 2,,” and missing spacing in author affiliations such as “Cartagena3” and “Marinaccio3”.
    The age inconsistency remains unresolved. The Abstract and Methods describe the athlete as 23 years old, whereas Table 1 reports 22 years. This should be harmonized.
    The Abstract still contains an awkward punctuation issue in the W4 sentence: “a 20 W increase in power at 4 mmol·L−1. [La−].” This should be corrected.
    Table 1 still reports TSH in ng·mL−1. Please verify the unit and reference range. The reference ranges for body composition variables and phase angle should also be supported or removed.
    Table 3 and the figure note still contain partly redundant information and inconsistent terminology, including the use of both “[La−]” and “Lactate.” Please harmonize terminology throughout.
    The informed consent statement should use “participant” or “athlete” rather than “patient.”
    The acknowledgment statement indicating that the authors “reviewed and edited the output” remains unclear. Please specify the nature of the output or tool used, in accordance with the journal’s editorial policy.
    The supplementary material link appears to contain a placeholder. This should be corrected before publication.
I hope these comments help you further strengthen the manuscript and support its publication potential.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The manuscript is generally readable, but it still requires targeted language editing to improve precision, consistency, and scientific tone. Several sentences remain overly assertive for a single-case design.
The authors should also correct residual grammatical, punctuation, formatting, and terminology inconsistencies