Fatiguing effects of indirect vibration stimulation in upper limb muscles: pre, post and during isometric contractions superimposed on upper limb vibration

Whole-body vibration and upper limb vibration (ULV) continue to gain popularity as exercise intervention for rehabilitation and sports applications. However, the fatiguing effects of indirect vibration stimulation are not yet fully understood. We investigated the effects of ULV stimulation superimposed on fatiguing isometric contractions using a purpose developed upper limb stimulation device. Thirteen healthy volunteers were exposed to both ULV superimposed to fatiguing isometric contractions (V) and isometric contractions alone Control (C). Both Vibration (V) and Control (C) exercises were performed at 80% of the maximum voluntary contractions. The stimulation used was 30 Hz frequency of 0.4 mm amplitude. Surface-electromyographic (EMG) activity of the Biceps Brachii, Triceps Brachii and Flexor Carpi Radialis were measured. EMG amplitude (EMGrms) and mean frequency (MEF) were computed to quantify muscle activity and fatigue levels. All muscles displayed significantly higher reduction in MEFs and a corresponding significant increase in EMGrms with the V than the Control, during fatiguing contractions (p < 0.05). Post vibration, all muscles showed higher levels of MEFs after recovery compared to the control. Our results show that near-maximal isometric fatiguing contractions superimposed on vibration stimulation lead to a higher rate of fatigue development compared to the isometric contraction alone in the upper limb muscles. Results also show higher manifestation of mechanical fatigue post treatment with vibration compared to the control. Vibration superimposed on isometric contraction not only seems to alter the neuromuscular function during fatiguing efforts by inducing higher neuromuscular load but also post vibration treatment.


Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? Yes

Is the language acceptable? No
Is it clear how to access all supporting data? Not Applicable

Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
In the current manuscript, authors have tried to understand the mechanism of neuromuscular fatigue for upper limb during isometric exercise with and without vibration stimulation. The topic of research is worthwhile and would increase our current understanding of vibration stimulation at a higher tension level of muscles during the task for the upper limb. In general, the study is methodology is explained well but the presentation of results and stats in its current form fails to succinctly represent the outcome, It needs rewriting.
I would like that author to address/consider the following points to improve the presentation of the paper 1) Please cite some literature which talks about the decrease in MEF as a measure of fatigue in the introduction. 2) Result section has a lot of emphasis on subjective/trends, which can be just part of just normal variance when you record measure like MVC, please only present the significant results mainly and trends can be mentioned in very brief and that too in discussion not in the results section.
3) There are too many figures, maybe authors can combine them in subplots to improve readability, also use the asterisk, where necessary to show significant differences as authors have done in later figures -Maybe an author can consider presenting %changes 4) Please avoid the explanation of the results in the results section like following lines, page 7 lines 36-41,45-48,51-53 page 9 lines 42-43 they need to be moved in the discussion section. 5) section "EMG Amplitude Results-Pre and Post Fatigue Exercise-EMGrms" needs to be completely rewritten after reading it number of times, it's not clear what's happening, again please do not overemphasize on trends and focus on statistically significant results 5) Table 4 have P values <0.05 but they are not marked as significant, please check and explain 6) in "EMG Frequency Results-During Fatigue Exercise-Mean Frequency (MEF)" section authors are talking about slopes, but not an objective measure of the slope is discussed in methods section or even in results, authors are merely describing the trends visible from figures. Authors can present % change in slopes. Again, please refrain from the explanation of results page 10 line 53-56, line 57-60. Page 12 line 1-11. In this section the number of figures can be reduced, authors can consider using subplots. 7) It's hard to conclude anything from the presented results a lot more focus is required in presenting the results. Because results are obscure so its hard to read the discussion section. Once the Results section is revamped based on above comments, hopefully, it will easier to read the discussion and make sense of the work presented. 8) Journal name is missing from the following citation, please double check all the references -Mischi M, Cardinale M Muscle electrical activity during force modulation exercise. vol. 2008 pp. 2065-2068. Review form: Reviewer 3

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? Yes

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
The manuscript presents an interesting study on the application of vibration exercise to the upper-limb muscles in order to evaluate fatigue. The results show, as expected, increased myoelectric fatigue with vibration exercise, with the exception of an unexpected increase in the EMG mean frequency during the MVC test post exercise. The manuscript is clear and well written.
Hereafter I is the list of my comments and remarks.
Page 4: the concept of "indirect vibration", as opposed to direct vibration, should be explained in the introduction as not all readers are familiar with this terminology.
Page 4, line 54: I cannot find in the manuscript results "post recovery" but only before and after the exercises. Please explain.
Page 7: it would be useful to the reader to have a figure showing the exact electrode positioning, with respect to the fiber orientation as well as to the muscle innervation.
Page 8, EMG data processing: have the authors applied a band-pass filter (e.g., 20-400 Hz) before the analysis? Low-frequency noise (<20 Hz) could explain the increase in MEF obtained after vibration exercise.
Page 9, Statistical analysis: It is unclear how the different segments of each recording are compared in the statistics since they are all of different length and timing, depending on the subject and the trial (which stops at 60% of the MVC). This also refers to the plots in Figs 10-17. It seems that the values from measurements 1 to 5 are not necessarily referring to the same time. The authors should elaborate on this.
Page 9, Statistical analysis: is the use of a t-test motivated by a Gaussian distribution of the data?
In the fatigue plots, Figs 10-17, the authors may also like to consider the decay (fatiguing rate), taken as the angular coefficient of the linear fit to the points. This should differ for vibration and control.

19-Jun-2019
Dear Dr Pujari, The editors assigned to your paper ("Fatiguing Effects of Indirect Vibration Stimulation in Upper Limb Muscles-post and during Isometric Contractions Superimposed on Upper Limb Vibration") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
Please submit a copy of your revised paper before 12-Jul-2019. Please note that the revision deadline will expire at 00.00am on this date. If we do not hear from you within this time then it will be assumed that the paper has been withdrawn. In exceptional circumstances, extensions may be possible if agreed with the Editorial Office in advance. We do not allow multiple rounds of revision so we urge you to make every effort to fully address all of the comments at this stage. If deemed necessary by the Editors, your manuscript will be sent back to one or more of the original reviewers for assessment. If the original reviewers are not available, we may invite new reviewers.
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All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. The expert reviewers have made a number of suggestions for revising this paper to more clearly convey the specific reasoning and hypotheses of the study, and to more clearly and concisely present the results. Addressing these comments will require substantial revision to the paper, and possibly another round a review.
When making the revisions, please particularly focus on making the message of the paper specific, focused and clear. Please try to consolidate the figures and results to focus on presenting the data that is directly needed. Seventeen figures seems like too many, and the data are currently presented in a format that wastes a lot of ink and page space. For example, results for the same muscle across conditions might be consolidated into a single multi-panel figure with unnecessary gridlines and box outlines removed. Consider organizing and consolidating the figures in a way that most directly conveys the key findings required test the main hypotheses of the paper.

Comments to Author:
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) Significance The authors investigate an interesting topic regarding the neuromuscular response in the upper limb muscles by superimposing indirect localized vibration to a defined level of isometric muscle contraction (by using a new device). Findings of this study suggest that near maximal isometric fatiguing contractions superimposed on vibration stimulation lead to a higher rate of fatigue development compared to the isometric contraction alone. Vibration superimposed on isometric contraction alters the neuromuscular function during fatiguing efforts and post vibration treatment, thus the results contribute to increase the body of knowledge.
Accuracy of title and abstract Title and abstract of the manuscript are adequate and provide an accurate understating of inquiry of the study.
Clarity of hypothesis and rationale Hypothesis should be more specific and underline the novelty of the present study; that is: the level of isometric muscle contraction is exactly defined by means of a new pulley system! Actually, the two hypothesis appear too general.

Adequacy of experimental design and methods
This section is clear and well detailed.

Quality of data and presentation of results
In the section results should be reported the p-values and the effect size. Tables and figures should be reorganized. In my opinion, the use of tables and figures to show the same results is not appropriate. The symbols indicating the significant differences should be included in the figures. In this way the reader is facilitated to understand the section results. Considering the large standard deviations, the Authors could report the standard errors in place of standard deviations. Please, check the symbols (indicating the statistical significance) in the table 3 and 4.
Length and appropriateness of discussion This section opens with the restated aim of the study which is consistent with the purposes expressed in the introduction. The physiological reasoning has been expanded and the limitations addressed. Page 18, lines 42-44. Please, include some references. Please, check the reference 42. In synthesis, references reflect the most relevant and recent articles in the area of the study.

Reviewer: 2
Comments to the Author(s) In the current manuscript, authors have tried to understand the mechanism of neuromuscular fatigue for upper limb during isometric exercise with and without vibration stimulation. The topic of research is worthwhile and would increase our current understanding of vibration stimulation at a higher tension level of muscles during the task for the upper limb. In general, the study is methodology is explained well but the presentation of results and stats in its current form fails to succinctly represent the outcome, It needs rewriting.
I would like that author to address/consider the following points to improve the presentation of the paper 1) Please cite some literature which talks about the decrease in MEF as a measure of fatigue in the introduction. 2) Result section has a lot of emphasis on subjective/trends, which can be just part of just normal variance when you record measure like MVC, please only present the significant results mainly and trends can be mentioned in very brief and that too in discussion not in the results section.
3) There are too many figures, maybe authors can combine them in subplots to improve readability, also use the asterisk, where necessary to show significant differences as authors have done in later figures -Maybe an author can consider presenting %changes 4) Please avoid the explanation of the results in the results section like following lines, page 7 lines 36-41,45-48,51-53 page 9 lines 42-43 they need to be moved in the discussion section. 5) section "EMG Amplitude Results-Pre and Post Fatigue Exercise-EMGrms" needs to be completely rewritten after reading it number of times, it's not clear what's happening, again please do not overemphasize on trends and focus on statistically significant results 5) Table 4 have P values <0.05 but they are not marked as significant, please check and explain 6) in "EMG Frequency Results-During Fatigue Exercise-Mean Frequency (MEF)" section authors are talking about slopes, but not an objective measure of the slope is discussed in methods section or even in results, authors are merely describing the trends visible from figures. Authors can present % change in slopes. Again, please refrain from the explanation of results page 10 line 53-56, line 57-60. Page 12 line 1-11. In this section the number of figures can be reduced, authors can consider using subplots. 7) It's hard to conclude anything from the presented results a lot more focus is required in presenting the results. Because results are obscure so its hard to read the discussion section. Once the Results section is revamped based on above comments, hopefully, it will easier to read the discussion and make sense of the work presented. Comments to the Author(s) The manuscript presents an interesting study on the application of vibration exercise to the upper-limb muscles in order to evaluate fatigue. The results show, as expected, increased myoelectric fatigue with vibration exercise, with the exception of an unexpected increase in the EMG mean frequency during the MVC test post exercise. The manuscript is clear and well written.
Hereafter I is the list of my comments and remarks.
Page 4: the concept of "indirect vibration", as opposed to direct vibration, should be explained in the introduction as not all readers are familiar with this terminology.
Page 4, line 54: I cannot find in the manuscript results "post recovery" but only before and after the exercises. Please explain.
Page 7: it would be useful to the reader to have a figure showing the exact electrode positioning, with respect to the fiber orientation as well as to the muscle innervation.
Page 8, EMG data processing: have the authors applied a band-pass filter (e.g., 20-400 Hz) before the analysis? Low-frequency noise (<20 Hz) could explain the increase in MEF obtained after vibration exercise.
Page 9, Statistical analysis: It is unclear how the different segments of each recording are compared in the statistics since they are all of different length and timing, depending on the subject and the trial (which stops at 60% of the MVC). This also refers to the plots in Figs 10-17. It seems that the values from measurements 1 to 5 are not necessarily referring to the same time. The authors should elaborate on this.
Page 9, Statistical analysis: is the use of a t-test motivated by a Gaussian distribution of the data?
In the fatigue plots, Figs 10-17, the authors may also like to consider the decay (fatiguing rate), taken as the angular coefficient of the linear fit to the points. This should differ for vibration and control.

Author's Response to Decision Letter for (RSOS-190019.R0)
See Appendix A.

Recommendation?
Accept as is Comments to the Author(s) Thanks for a thorough revision of the manuscript. In my opinion the text has improved considerably and it is ready for publication.

Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Recommendation?
Accept as is You can expect to receive a proof of your article in the near future. Please contact the editorial office (openscience_proofs@royalsociety.org and openscience@royalsociety.org) to let us know if you are likely to be away from e-mail contact --if you are going to be away, please nominate a coauthor (if available) to manage the proofing process, and ensure they are copied into your email to the journal.
Due to rapid publication and an extremely tight schedule, if comments are not received, your paper may experience a delay in publication.
Royal Society Open Science operates under a continuous publication model (http://bit.ly/cpFAQ). Your article will be published straight into the next open issue and this will be the final version of the paper. As such, it can be cited immediately by other researchers. As the issue version of your paper will be the only version to be published I would advise you to check your proofs thoroughly as changes cannot be made once the paper is published. To analyse the effect of treatment (Control Vs Vibration) and effort (MVC1 to MVC4), we have now employed 2 way Anova and the results comparing EMGrms means between effort levels (MVC1 to MVC4) and between control and vibration are now included in Table 1 and 2 in the manuscript.

Comment 7:
In EMG Frequency Results-During Fatigue Exercise-Mean Frequency (MEF)" section authors are talking about slopes, but not an objective measure of the slope is discussed in methods section or even in results, authors are merely describing the trends visible from figures. Authors can present % change in slopes. Again, please refrain from the explanation of results page 10 line 53-56, line 57-60. Page 12 line 1-11. In this section the number of figures can be reduced, authors can consider using subplots.
Response: Agree with this comment, an objective measure quantifying the slopes was not included. We have now quantified the reduction in MEF values i.e. changes in the slope with regression analysis (using least square method). Now all the fatigue figures include this quantified measure of change in slope, which now has been used to support our discussion. Explanation of results has been moved to discussion section. We have tried reducing the number of figures throughout the manuscript. Bar graphs in the results have now been combined to improve readability. However, due to the nature of the fatigue figures, combining them as subplots makes the figure illegible. Overlaying fatigue graphs is also not an option as the trends of the plots are similar; they make the graph lines obscure when overlaid on one another. Hence the fatigue figures have been kept similar however, each fatigue figure now includes quantified measure of the change of slope.
Comment 8: It's hard to conclude anything from the presented results a lot more focus is required in presenting the results. Because results are obscure so it's hard to read the discussion section. Once the Results section is revamped based on above comments, hopefully, it will easier to read the discussion and make sense of the work presented.
Response: Results section has now been improved to convey the message more clearly and hopefully reads better. To summaries, among others, following changes have been carried out to improve the focus and readability. 1) Explanation of results has been avoided in the results section and moved to discussion section. 2) Figures with bar graphs have been combined with statistical significant values clearly indicated. 3) Objective measure of slope change is now included to support the discussion. 4) Less emphasis is placed on trends. Response: Reference has been corrected and all the references have been rechecked for their details.

Reviewer 3:
Comment Page 4: the concept of "indirect vibration", as opposed to direct vibration, should be explained in the introduction as not all readers are familiar with this terminology.