Slow-Paced Breathing: Influence of Inhalation/Exhalation Ratio and of Respiratory Pauses on Cardiac Vagal Activity

Round 1

Reviewer 1 Report

Title: as a suggestion, please consider the possibility to change “resting-periods” in “breathing pauses”, as “resting-period” may be misinterpreted as “resting-state” just by reading the title

Abstract - Line 25: please include more information on the breathing conditions, such as the duration, the calculated I/E ratios, and the lengths of the breath pauses

Abstract - Line 27: as a suggestion, maybe it is better to change “post-inhalation and post-exhalation resting period” with “post-inhalation and post-exhalation pauses” throughout the paper

Line 35-36 contain the repetition “cope with stress”

Line 40: include a brief definition of the resting periods (or pauses) between respiratory phases

Line 97-98: please include and describe also the work from Edmonds et al., 2009 “A Single-Participants Investigation of the Effects of Various Biofeedback-Assisted Breathing Patterns on Heart Rate Variability: A Practitioner’s Approach”

Line 103: explain briefly that 10 cpm cannot be considered SPB

Line 122: how long was the post-exhalation rest period?

Line 145: can you please better explain why to choose athletes and not people from the general population? It is not really clear from the manuscript

Line 180: please better describe the characteristics of each breathing conditions also in the main text, for example the actual I/E ratio and the length of each breathing phase

Important: the variations of I/E ratio are very little in the present study, as they range from 0.8 to 1.2. Why the authors chose to use these little differences, for example comparing them with I/E ratios investigated by Edmonds et al., 2009 (range: 0.5 - 1), Van Diest et al., 2014 (range: 0.42 – 2.33), and Strauss-Blasche et al., 2000 (range: 1 - 3.4). Please include the motivation in the discussion and eventually a brief paragraph in the limitations section

Line 186 – Figure 1: the figure is of very bad quality and unesthetic, in order to be suitable for publication in “sustainability”, please enhance the quality of all the arrows and all the panels. Include also the unit of measure, the direction of inhalation (“is up”) and exhalation (“is down”), and make a connection between the horizontal little black arrows and the respective breath phase

Line 200: please better describe the “5-min rest period”. Maybe it is better to call it "baseline", and describe its characteristics such as the spontaneous breathing, the open eyes, and eventually that it was characterized by relaxation and mind-wandering. Also, include it in Figure 2

Line 207 – Figure 2: as for Figure 1, Figure 2 needs a complete restyling in order to be suitable for publication

Line 217: corrected alpha is 0.008, not 0.08

Line 218-219: please add more details about statistical testing, if it was two-tailed or not etc

Line 230-238: please display significant results in a new figure OR in a new table, highlighting statistically significant results with an *

Line 289: please expand the Limitation section. You should refer to the choice of athletes instead of participants from the general population, and consider the possibility of different result in those participants. Moreover, consider the possibility that 5-minutes of washout between the conditions were not enough to cancel the effect of previous breathing techniques. Finally, as an important limitation, discuss the possibility that participants may have not followed completely or partially the breathing exercises. It seems that without a respiratory belt it is not possible to evaluate if the participants performed the exercise or not. The authors said that they used Kubios for HRV analysis. To my knowledge, it is possible to reconstruct the breathing signal from the ECG with some Kubios functions. Please consider the possibility to do that, or alternatively to include this issue as an important limitation

Line 292-296: please delete these lines as they seem redundant and unnecessary

Author Response

Reviewer 1

First of all, we would like to thank Reviewer 1 for taking the time to review our manuscript, and providing helpful comments. Please find our response to the comments below. The manuscript has also been submitted with corrections highlighted in red. We are pleased that Reviewer 1 saw merit in the study. We believe that by addressing their concerns, we have substantially strengthened the final manuscript.

Title: as a suggestion, please consider the possibility to change “resting-periods” in “breathing pauses”, as “resting-period” may be misinterpreted as “resting-state” just by reading the title

Thanks for pointing this out. The rationale for our initial choice was that Russell et al. (2017) used this term, but we understand the possible confusion. So we agree with the suggestion, and now changed “resting period” to “respiratory pause” in the title and throughout the manuscript.

Abstract - Line 25: please include more information on the breathing conditions, such as the duration, the calculated I/E ratios, and the lengths of the breath pauses

We added the following information to the abstract: “Each condition lasted 5min, with 30 respiratory cycles, each respiratory cycle lasted 10s (6 cycles per minute), with inhalation/exhalation ratios of 0.8, 1, 1.2; and with or without respiratory pauses (0.4s) between respiratory phases.” (p.1, l.26-28)

Abstract - Line 27: as a suggestion, maybe it is better to change “post-inhalation and post-exhalation resting period” with “post-inhalation and post-exhalation pauses” throughout the paper

According to your comment above, we changed any occurrence of “resting period” to “respiratory pause”.

Line 35-36 contain the repetition “cope with stress”

We changed accordingly to this comment.

Line 40: include a brief definition of the resting periods (or pauses) between respiratory phases

We now added a brief definition of respiratory pause: “and the presence of a respiratory pause (i.e. brief cessation of air flow) between respiratory phases”. (p.1, l.42)

Line 97-98: please include and describe also the work from Edmonds et al., 2009 “A Single-Participants Investigation of the Effects of Various Biofeedback-Assisted Breathing Patterns on Heart Rate Variability: A Practitioner’s Approach”

Thanks for bringing this relevant paper to our attention, we now mention it at several places in the manuscript:

“Additionally, biofeedback can assist in identifying the breathing pattern which leads to the highest CVA increase (Edmonds et al., 2009; Lehrer & Gevirtz, 2014; Lehrer et al., 2000; Shaffer & Meehan, 2020).” (p.2, l.97-98)

“Finally, Edmonds et al. (2009) investigated the influence of different breathing patterns around 6cpm: 1:1 breathing ratio with post-inhalation and post-exhalation respiratory pauses, 1:1 ratio with no respiratory pauses, 1:2 breathing ratio with no respiratory pauses, 1:2 with post-inhalation and post-exhalation respiratory pauses, and finally a condition requiring the participants to breathe in phase with heart rate. The focus was not on between-subject analysis, but on within-subject analysis, and the authors found that for each participant, a specific breathing pattern produced the highest increase in LF. Moreover, all breathing patterns were found to produce a descriptive increase in LF, however no inferential statistics were run at the group level to investigate differences between breathing patterns.” (p.3, l.137-146)

Line 103: explain briefly that 10 cpm cannot be considered SPB

While spontaneous breathing is comprised between 12 and 20 cpm (Sherwood, 2006; Tortora & Derrickson, 2014), slow-paced breathing was suggested to have an upper limit at 10 cpm in a recent systematic review (Zaccaro et al., 2018).

We added: “In addition, Strauss-Blasche et al. (2000) did not focus on SPB (10 cpm), SPB being characterized by breathing frequencies lower than 10 cpm (Zaccaro et al., 2018).” (p.3, l.128-129)

Line 122: how long was the post-exhalation rest period?

The resting period in Russell et al. 2017 was 4s, we now report it (p.4, l.157).

Line 145: can you please better explain why to choose athletes and not people from the general population? It is not really clear from the manuscript

To put things into context, this study was aimed to answer the call for the Special Issue: “Heart Rate Variability in Sustainable Health and Sport Contexts: New Insights and Perspectives »”, which explains the focus on an athletic sample.

We further provide the following rationale: “Athletes were here defined as individuals regularly engaging in sport training. Recruiting a homogenous athletic sample helps to limit inter-individual differences in HRV that can be found in the general population, enabling a better interpretation of the findings (Quintana & Heathers, 2014).” (p.4, l.192-195)

We also acknowledge that this is at the same time a limitation of the study, which limits generalization of the results, and we now indicate it as well in the Limitation section.

“Limitations include first the sample chosen, comprising only athletes. This limits the generalization of our findings, given athletes have higher resting HRV than the general population (Aubert et al., 2003), and a ceiling effect may appear with SPB. Future research has to investigate this research question in different samples.” (p.11, l.367-369)

Line 180: please better describe the characteristics of each breathing conditions also in the main text, for example the actual I/E ratio and the length of each breathing phase

We now describe the characteristics of each breathing condition in the text: “The six conditions were the following: 4.6s / 0.4s / 4.6s / 0.4s (inhalation/exhalation ratio = 1); 4.1s / 0.4s / 5.1s / 0.4s (inhalation/exhalation ratio = 0.8); 5.1s / 0.4s / 4.1s / 0.4s (inhalation/exhalation ratio = 1.2); 5s / 5s (inhalation/exhalation ratio = 1.0); 4.5s / 5.5s (inhalation/exhalation ratio = 0.8); 5.5s / 4.5s (inhalation/exhalation ratio = 1.2).” (p.4, l.230-234)

Important: the variations of I/E ratio are very little in the present study, as they range from 0.8 to 1.2. Why the authors chose to use these little differences, for example comparing them with I/E ratios investigated by Edmonds et al., 2009 (range: 0.5 - 1), Van Diest et al., 2014 (range: 0.42 – 2.33), and Strauss-Blasche et al., 2000 (range: 1 - 3.4). Please include the motivation in the discussion and eventually a brief paragraph in the limitations section

We now added to the Slow-paced breathing section: “The inhalation / exhalation ratio was 4.5s / 5.5s and 5.5s / 4.5s, based on Allen and Friedman (2012).” (p.5, l.230-231)

We now added this point as a limitation of our study:

“Additionally, inhalation/exhalation ratio range was reduced (0.8-1.2); and future research should consider investigating larger ranges (Edmonds et al., 2009; Strauss-Blasche et al., 2000; Van Diest et al., 2014).” (p.11, l.390-391)

Line 186 – Figure 1: the figure is of very bad quality and unaesthetic, in order to be suitable for publication in “sustainability”, please enhance the quality of all the arrows and all the panels. Include also the unit of measure, the direction of inhalation (“is up”) and exhalation (“is down”), and make a connection between the horizontal little black arrows and the respective breath phase

Thanks for this comment, we agree with the reviewer that the quality of the figures could be improved, and we now provided a revised version of Figure 1.

Line 200: please better describe the “5-min rest period”. Maybe it is better to call it "baseline", and describe its characteristics such as the spontaneous breathing, the open eyes, and eventually that it was characterized by relaxation and mind-wandering. Also, include it in Figure 2

We now added details of the 5-min rest period, and included them in Figure 2.

“The participants started with a 5-min rest period (baseline), where they were breathing spontaneously, with open eyes.” (p.6, l.255)

“The washout period characteristics were similar to those of the baseline.” (p.6, l.258-259)

Line 207 – Figure 2: as for Figure 1, Figure 2 needs a complete restyling in order to be suitable for publication

Thanks for this comment, similar as for Figure 1, we agree with the reviewer that the quality of the figures could be improved, and we now provided a revised version of Figure 2.

Line 217: corrected alpha is 0.008, not 0.08

Thanks for spotting this, corrected.

Line 218-219: please add more details about statistical testing, if it was two-tailed or not etc

We now added the information that the series of t-tests was two-tailed. (p.7, l.275)

Line 230-238: please display significant results in a new figure OR in a new table, highlighting statistically significant results with an *

We now displayed the significant results in Figure 3.

Line 289: please expand the Limitation section. You should refer to the choice of athletes instead of participants from the general population, and consider the possibility of different result in those participants. Moreover, consider the possibility that 5-minutes of washout between the conditions were not enough to cancel the effect of previous breathing techniques. Finally, as an important limitation, discuss the possibility that participants may have not followed completely or partially the breathing exercises. It seems that without a respiratory belt it is not possible to evaluate if the participants performed the exercise or not. The authors said that they used Kubios for HRV analysis. To my knowledge, it is possible to reconstruct the breathing signal from the ECG with some Kubios functions. Please consider the possibility to do that, or alternatively to include this issue as an important limitation.

We completed the Limitation section with the recommendation of the reviewer:

“This limits the generalization of our findings, given athletes have higher resting HRV than the general population (Aubert et al., 2003), and a ceiling effect may appear with SPB. Future research has to investigate this research question in different samples.” (p.11, l.367-369)

“Second, the 5-min washout period between the conditions might not have been enough to cancel the effects of previous breathing techniques.” (p.11, l.370-372)

Regarding the issue of following accurately the breathing technique, we added the following points:

We now added the calculation of breathing frequency, as calculated post-hoc with Kubios, as a manipulation check:

“Respiratory frequency was computed via the ECG derived respiration algorithm of Kubios (Tarvainen et al., 2014).” (p.5, l.218-219)

We added the descriptive statistics regarding breathing frequency in Table 1.

We added in the Results section: “The first manipulation check revealed that participants followed the 6-cpm breathing frequency, ranging from 6.48 (SD = 0.20) to 6.55 (SD = 0.26). A repeated-measures ANOVA with Greenhouse-Geisser correction was conducted, and showed no significant effect of condition on breathing frequency, with F(4.401, 277.232) = 0.696, p = .608, partial η2  = 0.01.” (p.7, l.281-285)

in the Limitation section: “Third, our equipment did not allow us to control precisely the exact duration of the inhalation, exhalation, and respiratory pauses, so it is not possible for us to evaluate how accurately the participants performed the breathing techniques. Still, we were able to control for respiratory frequency, to assure that our participants followed the 6 cpm rhythm, by using the respiration algorithm of Kubios (Tarvainen et al., 2014). Additionally, the experimenter paid close attention that the participants were following the breathing pacer. Finally, we also checked the visual display of R-R values with Kubios, given during slow-paced breathing oscillations matching the respiratory frequency can be observed (Lehrer & Gevirtz, 2014). Nonetheless, future research should use a respiratory belt to offer an online measurement of respiratory frequency.” (p.11, l.372-381)

Line 292-296: please delete these lines as they seem redundant and unnecessary

We now deleted those lines.

Reviewer 2 Report

The current study investigates the effect of inhalation/exhalation ratio and respiratory pauses (combined inspiratory and expiratory) on HRV in athletes. The introduction very nicely explains a broader context of the research question, discussing previous research studies in a fair and insightful way. However, I would like to present the following questions and suggestions to the authors.

Questions regarding methods:

• In the participants section, no information is provided on what the authors consider athletes. Also no information is available on recruitment methods. In addition, some demographics would be informative to better understand the sample studied, especially in light of inter-individual variability in HRV.
• What was the reasoning of the authors to impose a 4.5/5.5 or 5.5/4.5 inhalation/exhalation ratio? A 4/6 ratio is frequently used. Other studies cited by the authors have used a 3/7 ratio. What were the arguments to deviate from that?
• The authors discuss they performed a manipulation check. More specifically, they consider comparing RMSSD between the breathing conditions and baseline as a manipulation check.
  • To my opinion, this is not a manipulation check. Comparing RMSSD between the breathing conditions and baseline is a result in itself. A manipulation check would involve checking whether the measured breathing patterns matched the paced, imposed breathing patterns. Were inspiratory/expiratory time measured, or at least checked, in any way?
  • I also wonder what the baseline (as mentioned in the manipulation check) refers to in this study? If I am not mistaken, in the study design (including figure 2) only a familiarization phase and washout periods are mentioned.

Questions regarding results:

• Post-hoc tests following a main effect of inhalation/exhalation ratio are conducted. The authors discuss differences between inhalation < exhalation and the two other conditions. Were differences between inhalation > exhalation and inhalation = exhalation statistically significant?

Questions regarding discussion:

• A lack of significant effects is interpreted as the absence of effects rather than lack of evidence for an effect (e.g. CVA was not influenced by the presence of a resting period after the inhalation or exhalation phase).
• In the introduction and discussion, the authors suggest that “longer exhalation provokes a higher activation of the parasympathetic nervous system, which is reflected in CVA”. I wonder whether there is any physiological mechanisms that supports this hypothesis. To my opinion, the increase in CVA as a result of longer exhalation could as well be due to a longer activation of the parasympathetic nervous system, resulting in more time to allow vagal activity to work and to lower HR.
• The authors compare their results with those of Russel et al. [68]. One interpretation that is not discussed is that Russel et al. compared a 5-5 ratio to a 4-2-4 ratio, and thus any differences between the two conditions might be due to longer exhalation time, rather than a post-exhalation pause/rest. Actually exhalation time in the Russel et al. study is longer than in the current study (6s vs. 5.5s). So in part, the findings of Russel et al. (regardless of the interpretation in the Russel et al. paper) are consistent with the current findings.
• In the discussion, the context of study, being a study in athletes specifically, is lost a bit. I wonder to what extent the study being a study in athletes is important in its broader context.

Minor comments:

Line 209 and 213: Data were

Author Response

Reviewer 2    

First of all, we would like to thank Reviewer 2 for taking the time to review our manuscript, and providing helpful comments. Please find our response to the comments below. The manuscript has also been submitted with corrections highlighted in red. We are pleased that Reviewer 2 saw merit in the study. We believe that by addressing their concerns, we have substantially strengthened the final manuscript.

The current study investigates the effect of inhalation/exhalation ratio and respiratory pauses (combined inspiratory and expiratory) on HRV in athletes. The introduction very nicely explains a broader context of the research question, discussing previous research studies in a fair and insightful way. However, I would like to present the following questions and suggestions to the authors.

Questions regarding methods:

• In the participants section, no information is provided on what the authors consider athletes. Also no information is available on recruitment methods. In addition, some demographics would be informative to better understand the sample studied, especially in light of inter-individual variability in HRV.

We now added: “Athletes were here defined as individuals regularly engaging in sport training.” (p.4, l.192)

Unfortunately, we did not collect further demographic information regarding the type of sport practiced, and we now added this as a limitation of the study.

“Additionally, demographics related to the sport practiced have not been collected.” (p.11, l.370)

We mentioned the available demographics in the Participants section: “64 athletes (27 female; M_Age = 22, age range = 18-30 years old; BMI: M=23.10, SD=2.16; Waist-to-hips ratio: M=0.80, SD=0.08; number of sport hours per week: M=7.5h; SD=3.2)”. (p.4, l.192-195)

Regarding the recruitment procedure, we indicate in the Procedure section: “Participants were recruited via flyers on the campus of the local university and via posts on social network groups linked to the local university.”

• What was the reasoning of the authors to impose a 4.5/5.5 or 5.5/4.5 inhalation/exhalation ratio? A 4/6 ratio is frequently used. Other studies cited by the authors have used a 3/7 ratio. What were the arguments to deviate from that?

We acknowledge that different ratios have been previously used, we based our rationale on the following study: “The inhalation / exhalation ratio was 4.5s / 5.5s and 5.5s / 4.5s, based on Allen and Friedman (2012).” (p.6, l.241-242)

• The authors discuss they performed a manipulation check. More specifically, they consider comparing RMSSD between the breathing conditions and baseline as a manipulation check. To my opinion, this is not a manipulation check. Comparing RMSSD between the breathing conditions and baseline is a result in itself.

This is a valid point, we agree with the reviewer, and now present the comparison between RMSSD at baseline and in the different conditions as a result in itself. (p.8, l.286-293)

• A manipulation check would involve checking whether the measured breathing patterns matched the paced, imposed breathing patterns.

Thanks for the suggestion, also mentioned by Reviewer 1. We now added this manipulation check, based on the respiration analysis algorithm of Kubios (Tarvainen et al., 2014).

“The first manipulation check revealed that participants followed the 6 cpm breathing frequency, ranging from 6.48 (SD = 0.20) to 6.55 (SD = 0.26). A repeated-measures ANOVA with Greenhouse-Geisser correction was conducted, and showed no significant effect of condition on breathing frequency, with F(4.401, 277.232) = 0.696, p = .608, partial η2  = 0.01.” (p.8, l.281-285)

• Were inspiratory/expiratory time measured, or at least checked, in any way?

No, and this is indeed a limitation of our study, that we now indicate in the Limitation section.

“Third, our equipment did not allow us to control precisely the exact duration of the inhalation, exhalation, and respiratory pauses, so it is not possible for us to evaluate how accurately the participants performed the breathing techniques. Still, we were able to control for respiratory frequency, to assure that our participants followed the 6 cpm rhythm, by using the respiration algorithm of Kubios (Tarvainen et al., 2014). Additionally, the experimenter paid close attention that the participants were following the breathing pacer. Finally, we also checked the visual display of R-R values with Kubios, given during slow-paced breathing oscillations matching the respiratory frequency can be observed (Lehrer & Gevirtz, 2014).” (p11, l.281-285)

• I also wonder what the baseline (as mentioned in the manipulation check) refers to in this study? If I am not mistaken, in the study design (including figure 2) only a familiarization phase and washout periods are mentioned.

Thanks for pointing this out, it was indeed unclear. We now clarify what the baseline was in the Procedure section, and mention it clearly in Figure 2.

Questions regarding results:

• Post-hoc tests following a main effect of inhalation/exhalation ratio are conducted. The authors discuss differences between inhalation < exhalation and the two other conditions. Were differences between inhalation > exhalation and inhalation = exhalation statistically significant?

We now added the following sentence to the Results section: “No differences were found between the condition inhalation > exhalation and inhalation = exhalation, with t(63) = 1.155, Cohen’s d = 0.14, p = 0.758.” (p.8, l.303-306)

Questions regarding discussion:

• A lack of significant effects is interpreted as the absence of effects rather than lack of evidence for an effect (e.g. CVA was not influenced by the presence of a resting period after the inhalation or exhalation phase).

We reformulated this sentence: “There was no evidence for CVA to be influenced by the presence of a respiratory pause after the inhalation or exhalation phase.” (p.10, l.326-327)

• In the introduction and discussion, the authors suggest that “longer exhalation provokes a higher activation of the parasympathetic nervous system, which is reflected in CVA”. I wonder whether there is any physiological mechanisms that supports this hypothesis. To my opinion, the increase in CVA as a result of longer exhalation could as well be due to a longeractivation of the parasympathetic nervous system, resulting in more time to allow vagal activity to work and to lower HR.

Thanks for bringing this to our attention. We now add the following explanation: “It is expected that at slow breathing frequencies, more acetylcholine is released during exhalation, due to its longer duration (Taylor et al., 2001). Due to the time constants of acetylcholine hydrolysis around 1.5 to 2.0 s (Baskerville et al., 1979; Eckberg & Eckberg, 1982), longer exhalation is suggested to provoke a summation of sinoatrial responses, and hence maximally inhibit sinoatrial node firing.” (p.10, l.337-341)

• The authors compare their results with those of Russel et al. [68]. One interpretation that is not discussed is that Russel et al. compared a 5-5 ratio to a 4-2-4 ratio, and thus any differences between the two conditions might be due to longer exhalation time, rather than a post-exhalation pause/rest. Actually exhalation time in the Russel et al. study is longer than in the current study (6s vs. 5.5s). So in part, the findings of Russel et al. (regardless of the interpretation in the Russel et al. paper) are consistent with the current findings.

We would here kindly disagree with the reviewer regarding the interpretation mentioned. In Russel et al., in the 4-2-4 ratio, the exhalation time is 2s, followed by a 4s respiratory pause. We are unsure whether the 2s exhalation + 4s respiratory pause can be summed up and considered as a 6s exhalation, given the different physiological mechanisms at stake during exhalation and respiratory pause (apnea).

• In the discussion, the context of study, being a study in athletes specifically, is lost a bit. I wonder to what extent the study being a study in athletes is important in its broader context.

To put things into context, this study was aimed to answer the call for the Special Issue: “Heart Rate Variability in Sustainable Health and Sport Contexts: New Insights and Perspectives”, which explains the focus on an athletic sample.

We further provide the following rationale: “Athletes were here defined as individuals regularly engaging in sport training. Recruiting a homogenous athletic sample helps to limit inter-individual differences in HRV that can be found in the general population, enabling a better interpretation of the findings (Quintana & Heathers, 2014).” (p.4, l.192-195)

We added as well a point in the Limitation section:

“Limitations include first the sample chosen, comprising only athletes. Limitations include first the sample chosen, comprising only athletes. This limits the generalization of our findings, given athletes have higher resting HRV than the general population (Aubert et al., 2003), and a ceiling effect may appear with SPB. Future research has to investigate this research question in different samples.” (p.11, l.365-369)

Minor comments:

Line 209 and 213: Data were

These words have been modified accordingly.

Reviewer 3 Report

Slow and deep breathing is one of the most widely used techniques to regulate the autonomic system in the body. The present manuscript is important as it addressed two of the parameters of the performance of SPB techniques: a) inspiratory to expiratory prolongation and b) post inspiratory or expiratory resting period. The findings will help to know which breathing phase to focus on during the slow, deep breathing, to make it most beneficial. However, there are some questions and concerns:

Questions to the authors:

1. In the section procedure: It was not clear to me whether the six different breathing conditions randomised or counterbalanced until I looked to the graph. I recommend adding this to the text as well.
2. Clearly, there was no measure of respiration in this study (which in my opinion, is a big limitation), so this is impossible to understand how well the participants complied to the paced breathing, and we could not be assured of proper breathing performance. I believe this should be addressed properly as a limitation to the study.
3. The time of 0.4 seconds as a pause/rest after inspiration or expiration cycle is very short and it may not be even noticeable by the participant. Although the authors accepted this as a limitation of the work, the abstract of the manuscript suggests to the reader that any rest after inhalation or exhalation has no effect on RMSSD. From abstract: “The presence of a post-inhalation and post-exhalation resting period did not further influence RMSSD.” I do recommend revising this sentence to a more conservative and cautious sentence rather than this.

Author Response

Reviewer 3

First of all, we would like to thank Reviewer 3 for taking the time to review our manuscript, and providing helpful comments. Please find our response to the comments below. The manuscript has also been submitted with corrections highlighted in red. We are pleased that Reviewer 3 saw merit in the study. We believe that by addressing their concerns, we have substantially strengthened the final manuscript.

Slow and deep breathing is one of the most widely used techniques to regulate the autonomic system in the body. The present manuscript is important as it addressed two of the parameters of the performance of SPB techniques: a) inspiratory to expiratory prolongation and b) post inspiratory or expiratory resting period. The findings will help to know which breathing phase to focus on during the slow, deep breathing, to make it most beneficial. However, there are some questions and concerns:

Questions to the authors:

1. In the section procedure: It was not clear to me whether the six different breathing conditions randomised or counterbalanced until I looked to the graph. I recommend adding this to the text as well.

Thanks for this comment, we now added the information to the Procedure section. (p.5, l.257)

2. Clearly, there was no measure of respiration in this study (which in my opinion, is a big limitation), so this is impossible to understand how well the participants complied to the paced breathing, and we could not be assured of proper breathing performance. I believe this should be addressed properly as a limitation to the study.

We agree with this comment, and added the following limitation. “Third, our equipment did not allow us to control precisely the exact duration of the inhalation, exhalation, and respiratory pauses, so it is not possible for us to evaluate how accurately the participants performed the breathing techniques. Still, we were able to control for respiratory frequency, to assure that our participants followed the 6 cpm rhythm, by using the respiration algorithm of Kubios (Tarvainen et al., 2014). Additionally, the experimenter paid close attention that the participants were following the breathing pacer. Finally, we also checked the visual display of R-R values with Kubios, given during slow-paced breathing oscillations matching the respiratory frequency can be observed (Lehrer & Gevirtz, 2014).” (p11, l.281-285)

2. The time of 0.4 seconds as a pause/rest after inspiration or expiration cycle is very short and it may not be even noticeable by the participant. Although the authors accepted this as a limitation of the work, the abstract of the manuscript suggests to the reader that any rest after inhalation or exhalation has no effect on RMSSD. From abstract: “The presence of a post-inhalation and post-exhalation resting period did not further influence RMSSD.” I do recommend revising this sentence to a more conservative and cautious sentence rather than this.

Thanks for pointing this out, we now revised the sentence: “The presence of a brief (0.4s) post-inhalation and post-exhalation breathing pause did not further influence RMSSD.” (p.1, l.29-30)

Round 2

Reviewer 1 Report

I am pleased to see that all indications have been properly followed. The paper has improved a lot and it is suitable for publication in the present form

Reviewer 3 Report

Dear Chief Editor,

All comments addressed and the manuscript revised accordingly. I have no further comments. Thanks and good luck!

Best wishes