Dear Dr. Ruiz-Blais,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jun 07 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
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If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols....

We look forward to receiving your revised manuscript.

Kind regards,

Assoc. Prof. Phakkharawat Sittiprapaporn, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: No

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The 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.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.-->

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

Reviewer #1: This study examines the neural and cardiorespiratory effects of toning and singing compared to matched slow breathing. EEG and ECG were used to measure brain and cardiac activity in participants. The results indicate that toning and singing led to increased alpha and theta oscillations, whereas matched slow breathing alone did not produce these effects. While slow breathing significantly increased HRV-RMSSD, suggesting vagal activation, vocalization did not, implying distinct neural and autonomic mechanisms. These findings suggest that relaxation induced by singing or vocalization is unlikely to be solely driven by modifications in breathing patterns.

Overall, the study is relevant. However, several issues in the analyses and results require further clarification and discussion:

Major Issues:

1. Please provide the rationale for the chosen sample size. Was a power analysis conducted to determine adequacy?

2. Given the repeated nature of the measurements, a repeated measures ANOVA would be more appropriate than a two-way ANOVA.

3. Statistical analyses should be included to identify differentially activated brain clusters between the vocalization and breathing conditions. A cluster-based permutation test might provide a clearer picture of localized neural effects.

4. Were the p-values reported (e.g., lines 268–270) Bonferroni corrected? If so, please specify the correction method used. If not, multiple comparisons should be properly controlled.

5. Several physiological measures are listed in Table 1, but not all are discussed in the main text. Please ensure consistency by either elaborating on all measures or justifying their omission.

6. Given the diverse physiological measures applied, some of which are interrelated, a principal component analysis (PCA) or clustering analysis could provide a more holistic view of how these variables interact.

7. The rationale behind Figures 8 and 9 is unclear. What specific hypotheses or findings do these figures aim to illustrate?

8. The discussion on vocalization-induced relaxation requires further clarification. Notably, singing and toning appear to increase HR and lower HRV, both indicative of higher arousal (stress). Attention is linked to increased alpha waves—how do these changes in brain oscillations relate to the observed cardiac responses in different arousal/attentional states?

9. How does motor control differ between matched breathing and vocalization (toning/singing)? A discussion on the biomechanical and neural bases of phonation would help clarify this aspect.

Minor Issues:

1. Abbreviations should be defined upon first use.

2. HRV Metrics Reporting: SDNN, LF, HF, and RMSSD were calculated, but only RMSSD results were presented. Please clarify why the other measures were omitted.

Reviewer #2: This manuscript explores the neural and autonomic effects of vocal practices (toning and slow singing) compared to matched slow-breathing conditions. While the topic is timely and the premise intriguing, the current study lacks sufficient methodological rigor and mechanistic clarity to substantiate the key claims. It suggests dissociations between vocalization-evoked cortical oscillations and breathing-evoked vagal tone changes, but does not convincingly demonstrate mechanistic causality or neural-autonomic integration. The separation of neural and autonomic effects is suggested, but the analyses remain primarily correlative, with substantial physiological and methodological gaps, and the physiological interpretations are largely speculative. The work would benefit from a tighter conceptual framing and additional experimental controls.

Major comments

- The study reports post-intervention increases in alpha and theta power, yet task-state EEG is excluded due to vocalization artifacts. Without neural data during the intervention, conclusions about mechanisms or entrainment effects remain speculative.

- Vocalization inherently involves sensorimotor feedback loops (auditory-motor coupling, efference copy, etc.), which are not considered. Without controlling for auditory feedback, it's impossible to separate proprioceptive vocal-motor activity from auditory-induced entrainment effects. No analysis of vocal acoustic features (pitch, loudness, variability) was conducted, yet these could modulate arousal and autonomic effects.

- The authors attributes alpha/theta increases to "relaxation" or "mindfulness-like" states without including subjective measures of affect, relaxation, or mental state at those timepoints. They infer cortical relaxation via post-intervention alpha and theta power increases, yet no temporal dynamics or task-state EEG are estimated. This limits any causal or even associative interpretation of these changes. Further, the observed alpha/theta increases could reflect post-task relaxation rather than direct effects of vocalization.

- The authors treat HRV and EEG as separate outputs, yet the study's stated goal was to explore neural-autonomic coupling—which was not tested directly (e.g., via phase-amplitude coupling, Granger causality, etc.).

- The paper speculates about medullary afferent relay (via NTS) modulating alpha power, but without any latency analysis, ERP components, or HR-locked EEG, this remains highly speculative.

Technical comments

- No behavioral or subjective data were collected to validate the hypothesized relaxation or attentional states associated with EEG changes.

- The matched-breathing condition uses visual feedback of prior breathing patterns, which introduces:

- Asynchronous entrainment

- Differential attentional load

- A lack of proprioceptive coupling present in real-time vocalization

- The conditions are not matched for motor output, auditory feedback, cognitive engagement, or emotional valence—all critical confounds.

- The authors describe theta increases during toning were anterior, and posterior theta during singing. However, these topographical patterns of alpha and theta are not studied and are not statistically compared in a meaningful way.

- Beta increases are noted across all conditions and linked to attention, but the interpretations are vague and speculative. The negative correlation with RMSSD is interesting but likely epiphenomenal without mechanistic support.

- The use of HRV-RMSSD as a proxy for vagal tone is correct in principle, but the discrepancy between SDNN/LF and RMSSD is not deeply analyzed.

- The assertion that vocalization suppresses vagal tone via HR increases is plausible but not directly demonstrated. Any of the following would be relevant:

- Beat-to-beat coupling or cardio-vagal coherence

- Time-resolved measures across the intervention

- Simultaneous blood pressure/baroreflex data

- Artifact removal uses standard ICA and interpolations, but:

- No quantification of remaining noise variance

- No channel-wise SNR metrics

- No head motion capture to verify that vocalization-induced artifact wasn’t residual

Recommendations

- Include or recover task-state EEG data using artifact-resistant approaches to examine real-time dynamics.

- Incorporate subjective measures of emotional or cognitive states to support interpretations of EEG changes.

- Control or model auditory and attentional components across vocal and breathing conditions.

- Use time-resolved analyses of EEG-HRV coupling or HR-locked ERPs to support claims of central-autonomic dissociation.

- Consider alternative explanations such as physical exertion, sensory feedback, or performance relief to account for post-intervention EEG changes.

Reviewer #3: Thank you for the opportunity to review this interesting and well-executed manuscript titled "Neural and cardiorespiratory responses in vocalization and slow breathing: contrasting brain and autonomic responses."

This study addresses an important and understudied question: to what extent the benefits of vocal practices such as toning or slow singing can be attributed to the vocalization itself as opposed to slow breathing patterns. By combining EEG and heart rate variability (HRV) measures in a well-controlled within-subject design, the authors provide compelling evidence for a dissociation between neural and autonomic effects. The findings — increased alpha and theta power following vocalization but increased RMSSD (vagal tone) only during slow breathing — are intriguing and contribute valuable insights to the psychophysiological mechanisms of contemplative and therapeutic practices.

Strengths of the manuscript:

The study is methodologically robust, using a 2x2 design to contrast vocalization and matched breathing conditions.

EEG preprocessing and statistical analysis (including cluster-based permutation testing) are rigorous and clearly described.

HRV reanalysis includes both traditional metrics (SDNN, LF) and RMSSD, which is more directly linked to vagal tone.

The discussion is nuanced and acknowledges both the theoretical implications and the methodological limitations.

The writing is clear, and the figures effectively illustrate the key results.

Areas for improvement:

Data availability: The current data availability statement (“available upon request”) does not meet PLOS ONE’s open data policy. To proceed toward publication, the authors should deposit the anonymized dataset in a public repository (e.g., OSF, Zenodo, Dryad) and include the DOI or permanent link in the manuscript.

Participant exclusion: Seven participants were excluded from the original sample of 23. While this is not unusual in EEG research, it would strengthen the manuscript to clarify the reasons and potential implications (e.g., on statistical power or sample representativeness).

Justification for exploratory analyses: The inclusion of beta and low-gamma EEG bands is labeled as exploratory, but the rationale for these choices could be more thoroughly discussed or contextualized within the literature.

Timeline clarity: A visual schematic of the experimental protocol and timing (baseline, intervention, post-intervention, etc.) would improve accessibility, especially for interdisciplinary readers.

Language and phrasing: The manuscript is well written, but a few minor edits would improve clarity (e.g., rephrasing “HRV was greater for breathing-only” to “HRV was higher during the breathing-only condition”).

Ethical considerations:

The manuscript meets ethical standards. The study received IRB approval (certificate number 30004786), informed consent was obtained from all participants, and the procedures follow the Declaration of Helsinki.

Conclusion:

This is a solid and insightful contribution to the field. I recommend minor revisions to improve transparency, enhance compliance with data policies, and further strengthen the clarity of reporting. Once these issues are addressed, the manuscript should be suitable for publication in PLOS ONE.

Thank you again for the opportunity to review this work.

**********

what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Peddy CaliariPeddy CaliariPeddy CaliariPeddy Caliari

**********

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.. Please note that Supporting Information files do not need this step.. Please note that Supporting Information files do not need this step.. Please note that Supporting Information files do not need this step.

Dear Dr. Ruiz-Blais,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Nov 03 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols....

We look forward to receiving your revised manuscript.

Kind regards,

Assoc. Prof. Phakkharawat Sittiprapaporn, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: No

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The 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.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.-->

Reviewer #2: No

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

Reviewer #3: Yes

**********

Reviewer #2: It is evident that the authors have rejected every single statement of me and the other reviewers and they have just opted to respond by rejecting the point or acknowledging it and refusing to modify anything in their manuscript.

As such, my suggestion is Rejection.

Reviewer #3: Thank you for submitting the revised version of your manuscript. The authors have taken into account the feedback from the first round of review and significantly improved the overall clarity and scientific rigor of the paper.

Strengths of the revised manuscript:

The tracked-changes version allows easy identification of the modifications made.

The introduction and theoretical rationale have been slightly reworded for clarity.

The authors have clarified methodological aspects that were previously unclear, especially regarding the HRV measures and breathing conditions.

The figures and statistical results are better presented, and the legends are now self-explanatory.

The authors have responded adequately to the comments raised regarding the interpretation of the findings and have softened some overly conclusive statements.

Language quality is satisfactory and meets the journal's standards.

Minor remaining suggestions:

A few sentences in the introduction and discussion could benefit from tighter phrasing to improve readability. However, this does not compromise the article’s intelligibility.

It may be helpful to further emphasize, in the conclusion, the differential effect between afferent vagal tone and alpha power, and what this might imply for therapeutic applications.

In conclusion, I find the revised manuscript acceptable for publication in PLOS ONE. I thank the authors for their careful and constructive revision.

**********

what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #2: No

Reviewer #3: Yes: Peddy CaliariPeddy CaliariPeddy CaliariPeddy Caliari

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.. Please note that Supporting Information files do not need this step.-->

Dear Dr. Ruiz-Blais,

Please submit your revised manuscript by Feb 21 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols....

We look forward to receiving your revised manuscript.

Kind regards,

Jenna Scaramanga

Staff Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The 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.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.-->

Reviewer #3: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #3: Yes

**********

Reviewer #3: The revised manuscript is clear, coherent, and scientifically solid. It makes a valuable contribution by characterising neural (EEG) and autonomic (HRV) responses associated with vocalisation (toning, slow singing) versus matched slow breathing.

The combination of cluster-based EEG spectral analyses and HRV indices including RMSSD, which is more appropriate than LF/SDNN at low breathing frequencies, is methodologically rigorous and well motivated. The addition of figures summarising the procedure and preprocessing pipeline considerably improves accessibility for readers who are not EEG specialists.

Importantly, in this revision you:

- clarify your actual aims as describing neural profiles and parasympathetic responses in parallel, rather than directly testing “neural–autonomic coupling”;

- acknowledge and integrate key limitations (absence of task-state EEG, lack of direct coupling analyses, no subjective measures in this paper, unmatched motor/auditory/emotional features between conditions);

- temper several mechanistic claims, now explicitly presented as speculative and grounded in prior literature rather than as conclusions from the current dataset.

Overall, the manuscript is now much improved and scientifically sound. My remaining comments are minor and mostly editorial.

Follow-up on previous comments

You now provide a clear and reasonable justification for focusing on pre/post resting baselines instead of task-state EEG, given the substantial and condition-specific motion artefacts during vocalisation. You appropriately frame this as a limitation and point to future work using more artefact-resistant approaches (e.g., HERPs or other time-resolved methods).

You also explicitly acknowledge that the present design does not allow you to disentangle auditory feedback from vocal-motor contributions, that subjective/phenomenological aspects are reported elsewhere (Snow et al., 2018), and that the matched breathing and vocal conditions are not perfectly matched in terms of motor output, auditory feedback, attentional load or emotional valence. These are now treated as limitations and future design directions, which is scientifically honest and consistent with the exploratory nature of the study.

Your revision of the Abstract (“describe neural responses and autonomic responses”) and clarification of the study goals in the Introduction are now consistent with the actual analyses, which treat EEG and HRV as parallel but separately analysed outcomes. The discussion of medullary relay, NTS and vagal mechanisms is now clearly presented as hypothetical and based on prior work rather than as mechanistic evidence from the current dataset.

Remaining minor points and suggestions

1. Female/male ratio vs. final sample size

Please correct the apparent inconsistency between the reported female/male ratio and the final sample size (N = 16). Either provide the sex breakdown for the final analysed sample, or clearly distinguish between the sex distribution of the initial recruited sample and that of the final dataset.

2. “Afferent” vs. “efferent” vagal tone in the Abstract

In the Abstract, RMSSD is described as indexing “afferent vagal tone”, whereas in the Methods it is presented more generally as a vagal HRV measure associated with parasympathetic cardiac control, typically interpreted as efferent vagal activity to the heart. I recommend revising the Abstract to refer to RMSSD as “vagal tone”, “vagal activity” or “parasympathetic cardiac control”, in line with the Methods and HRV guidelines.

3. Wording about discarding post-task relaxation

Your argument that condition-specific and topographical differences make a purely generic post-task relaxation explanation unlikely is reasonable. I would simply suggest softening the sentence where you “discard” this explanation, for example: “we consider a purely generic post-task relaxation explanation unlikely in light of the condition-specific and topographical patterns observed.”

4. Framing of mechanistic interpretations

You already mark mechanistic interpretations (NTS, baroreflex pathways, competing influences of motor arousal and breathing frequency, etc.) as hypothetical. I encourage you to maintain this careful wording throughout the Discussion, using formulations such as “it has been hypothesized that…”, “one possible explanation is…”, or “our findings are consistent with the idea that…”, rather than stronger causal language.

5. Clarifying the reporting scale for HRV values (optional)

If HRV values in the main tables are reported on the original (untransformed) scale, it could help readers if this is stated explicitly (e.g., “HRV values are reported on the original scale for interpretability, although analyses were conducted on transformed data”).

6. Effect sizes for key results (optional)

Reporting simple effect sizes (e.g., partial η² for main ANOVA factors, Cohen’s d for key contrasts) would further strengthen the statistical reporting and help readers gauge the magnitude of the observed effects, although I do not consider this a requirement for publication.

Overall, once these minor points are addressed, I would be pleased to see this work published in PLOS ONE.

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Reviewer #3: Yes: Peddy CaliariPeddy CaliariPeddy CaliariPeddy Caliari

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Neural and cardiorespiratory responses in vocalization and slow breathing: contrasting brain and autonomic responses

PONE-D-24-57770R3

Dear Dr. Ruiz-Blais,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.  I apologize for the long delay in processing the manuscript as I have been struggling with obtaining reviewers for your revision. After my careful reading of your work, I agree with the reviewer that the revisions are acceptable for publication.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available??>

The 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.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.-->

Reviewer #3: No

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #3: Yes

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Reviewer #3: Thank you for your revised submission and detailed point-by-point response. The main minor points raised in my previous review have been adequately addressed. In particular, the sex ratio has been corrected to match the final analysed sample, the wording around HRV-RMSSD has been clarified to avoid potentially misleading “afferent/efferent” phrasing, and the reporting has been strengthened by clarifying the HRV reporting scale and adding effect sizes (partial η² and Cohen’s d).

Overall, the manuscript is now clearly written, technically sound, and the conclusions are appropriately supported by the data. I have no further substantive comments and recommend acceptance.

Kind regards,

Peddy Caliari

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #3: Yes: Peddy CaliariPeddy CaliariPeddy CaliariPeddy Caliari

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