Electrical therapies and cryotherapy offer limited effect in the treatment of EIMD; however, inconsistencies in the dose and frequency of these and other interventions may account for the lack of consensus regarding their efficacy.

Both as a cause and a consequence of this, there are very few evidence-based guidelines for the application of many of these interventions. Conversely, there is unequivocal evidence that prior bouts of eccentric exercise provide a protective effect against subsequent bouts of potentially damaging exercise.

Further research is warranted to elucidate the most appropriate dose and frequency of interventions to attenuate EIMD and if these interventions attenuate the adaptation process. This will both clarify the efficacy of such strategies and provide guidelines for evidence-based practice.

Abstract Exercise-induced muscle damage EIMD can be caused by novel or unaccustomed exercise and results in a temporary decrease in muscle force production, a rise in passive tension, increased muscle soreness and swelling, and an increase in intramuscular proteins in blood.

A timed rest period of 3min took place between each set. Before performing each eccentric contraction, participants raised the weight using both legs, concentrically. Each eccentric contraction lasted 3—5s, during which participants resisted the load with the dominant leg from full knee extension to 90 degrees angle of knee flexion Vaile et al.

All participants completed all seven sets. Water was provided ad libitum every 15min. Magnitude of DOMS was quantified using a visual analogue scale VAS , and it was self-rated by participants on a point-validated VAS indicating on a horizontal line with anchor points from 0 no pain to 10 extreme pain; Carlsson, ; McCormack et al.

Participants were seated with both legs in passive 90 degrees of flexion during a wall squat. Participants then placed a mark at the point on the VAS corresponding to their perception of soreness on the quadriceps muscle.

Participants were blinded to the scores they had previously reported. Maximal voluntary isometric contraction leg strength of the quadriceps was assessed on KINEO dynamometer Globus Kineo , Italy.

Participants were seated upright and strapped into the dynamometer to limit excess motion. The chair was adjusted so that the leg pad was placed on the lower part of the tibialis anterior, and the pivot was located on the lateral epicondyle of the dominant leg. Maximal force was measured at an angle of 60 degrees leg extension.

The protocol consisted of three maximal isometric contractions with s recovery between each repetition. Following a 2-min rest period, participants employed maximal isometric force against the leg pad. Peak force was determined by the average of three maximal isometric contractions lasting 3—5s.

Verbal encouragement was given throughout each repetition. A 6ml vacutainer tube of venous blood was collected at each time point lithium-heparin; BD, Oxford, United Kingdom.

Circulating CK activity was measured using a clinical chemistry analyser Werfen ILab Aries, Italy. All samples and standards were analysed in duplicate. Plasma EVs were prepared from the individual plasma thawed on ice aliquots μl per individual from each participant, under the different conditions, using sequential centrifugation and ultracentrifugation according to previously standardised and described protocols and procedures Kosgodage et al.

This was then centrifuged for 20min at 3, g at 4°C, to remove apoptotic bodies and aggregates. Supernatants were then collected and ultra-centrifuged at , g at 4°C for 1h.

This resulted in EV-enriched pellets, which were resuspended each in μl DPBS and thereafter ultra-centrifuged again for 1h at , g , at 4°C. The final resulting EV pellets were resuspended each in μl of DPBS.

Plasma-EV quantification and size distribution profiles were established by NTA, based on Brownian motion of particles in suspension, using the NanoSight NS system Malvern, United Kingdom. The diluted EV samples were applied to the NanoSight NS Malvern Panalytical, United Kingdom , recording five repetitive reads, 60s each.

Particle numbers per frame were 40—60, camera settings were at level 10 for recording and for post-analysis the detection threshold was set at 5. Replicate histograms were generated from these videos using the NanoSight software 3.

Plasma EVs were further assessed by morphological analysis using TEM. EVs were resuspended in mM sodium cacodylate buffer pH 7. One drop ~3—5μl of the EV suspension was placed onto a grid, which held a carbon support film which had been previously glow discharged.

Following partial drying of the EV suspension, the sample was fixed for 1min at room temperature RT by placing the grid onto a drop of a fixative solution 2.

The grid was applied to the surface of three drops of distilled water for washing of the EV sample, removing excess water using a filter paper. TEM imaging of EVs was carried out with a JEOL JEM transmission electron microscope JEOL, Tokyo, Japan , which was operated at 80kV, using a magnification of 30,x to 60,x.

Recording of digital images was performed with an AMT XR60 CCD camera Deben, United Kingdom. Extracellular vesicles were assessed for the EV-specific markers CD63 and Flotillin-1 Flot-1 , using western blotting. The membranes were then washed for 4×10min TBS-T, and visualised, using enhanced chemiluminescence ECL, Amersham, United Kingdom in conjunction with the UVP BioDoc-ITTM System Thermo Fisher Scientific, United Kingdom.

Normal distribution of data was examined by QQ plot visual inspection. Exercise-induced changes in EV profiles, CK, and MVIC were analysed using a mixed model ANOVA with repeated measures [group younger, older ×time pre-, post-, at 1, 2, 24, 48, 72h post-EIMD ].

As an ordinal measure, Mann-Whitney U test was used to determine between group differences in DOMS. The EIMD effects on DOMS within-group were determined across time using Freidman ANOVA, and the Wilcoxon matched pairs signed ranks test was performed for post hoc analysis to test differences in this variable.

The relationship between EV profiles, and CK, MVIC, and DOMS were performed with Pearson correlation. Partial eta-squared η 2 p values were calculated as measures of effect size for mixed model ANOVA when necessary, and were considered small 0.

Values were expressed as mean±SEM for data from parametric tests, and as median and interquartile range for data from non-parametric tests.

All figures were generated in, and statistical analysis performed in GraphPad Prism Version 9. Participant characteristics are presented in Table 1. Besides age, participants were reasonably homogenous, with no differences noted between body fat [younger older Extracellular vesicle profile both modal size and particle concentration was quantified by NTA representative sample shown in Figure 2A and were characterised by Western blotting for EV surface markers CD63 and Flot-1; Figure 2B and TEM for morphology Figure 2C.

Pre-exercise circulating blood samples suggested that EV modal size did not differ between younger and older participants [younger older 2. Figure 2. Measurement of EV modal size and count in younger and older participants. A Representative example of nanoparticle tracking analysis NTA , SEM shown in red and mean in black line.

B Western blotting of human plasma extracellular vesicles EVs showing positive for Flot-1 and CD C Transmission electron microscopy TEM images of human plasma-EVs, showing EV morphology; scale bar indicates nm.

Horizontal line indicates group means. Post hoc testing suggests force significantly decreased immediately post-EIMD [pooled pre-MVIC, Figure 3. Exercise-induced muscle damage reduces muscle force and increases circulating creatine kinase CK concentrations independent of age.

Red shaded zones indicate SEM in A,C , and interquartile range in B , and black connected line indicates group means in A,C , and medians in B , values of p between timepoints as indicated. The scale brake indicates from hourly testing to h intervals.

Mann-Whitney U test showed no significant difference in DOMS between groups at any timepoint. Nevertheless, DOMS returned to pre-EIMD values by 72h post-EIMD in the younger group, but not in the older group Figure 3B. pooled post-CK Figure 4. Alterations in EV modal size and count with exercise, and EV correlations with muscle damage markers.

Red shaded zones indicate SEM and black connected line indicates group means. Open circles indicate younger, closed indicate older. No significant associations were noted between MVIC and either ΔMode or ΔCount Figure 4F , or DOMS and either ΔMode or ΔCount at any time point measured Figure 4G.

Exercise is associated with a number of immediate physiological responses. Circulating EVs can act as plasma-based biomarkers, reflecting physiological and pathophysiological conditions of the body Withrow et al. Thus, this study analysed EVs in blood plasma isolated during the acute phase of EIMD and during a recovery period of 72h in younger and older healthy, physically active male adults.

In this study, we show that a single bout of EIMD triggers apparent changes to EV concentration and size distribution profiles, but in trained older men there is no clear differences in this EV signature from that of younger men.

However, unlike prior studies on the effects of acute endurance exercise on EV release profiles, acute eccentric resistance exercise does not appear to predictably alter EV modal size or EV concentration. Furthermore, immediate changes in EV profiles as observed here may associate with later changes in biological markers of muscle damage, such as CK, as found in the current study.

No significant effect on EV profiles was observed in relation to age at pre-exercise values, with younger and older participants showing relatively homogeneous EV profile responses.

Nonetheless, older participants had lesser magnitude of CK response than their younger counterparts. Whilst the younger group showed a greater signal in CK response and returned to pre-exercise values by the end of the experimental period, suggesting a better resolution in recovery, the older group did not attain absolute values by the end of the recovery period.

Unexpectedly, both groups had similar recovery in leg strength changes following EIMD. Likewise, a previous study has reported no age differences in muscle function after muscle-damaging exercise Heckel et al.

However, others concluded that younger individuals were able to recover and adapt quicker in functionality following EIMD, confirming that muscle function declines through the ageing process Tieland et al.

In the current study, muscle soreness significantly peaked immediately post- and at 24h post-EIMD for both groups, but the younger group consistently scored higher on perception of pain than the older group during the experimental period.

This may have been attributed to a higher muscle damage, as indicated by the increased CK activity for the younger men, or hypothetically due to the larger ratio of type II fibres typically seen in younger individuals, which have been suggested to be more susceptible to injury Byrne et al.

However, muscle biopsies would be required to confirm the fibre type shift. Similarly, Lavender and Nosaka reported older males experienced lower muscle soreness than younger males following EIMD. A review by Gibson and Helme also reported that pain perception is decreased with ageing.

This may explain the lower DOMS score of the older group compared with the younger group in the present study. Nevertheless, no significant differences were observed between groups following EIMD. Similarly, Nikolaidis and Heckel et al. However, Lavender and Nosaka found opposite findings after eccentric exercise.

The contrast in research findings was attributed to the magnitude of muscle damage induced by the exercise protocol used bilateral vs. unilateral or due to the different muscle group arm vs.

leg involved in the studies. Overall, the current study showed that EIMD recovery took a similar course in both muscle function and DOMS for physically active younger and older individuals.

Therefore, the data presented here suggests that when younger and older individuals are matched for activity status, ageing does not appear to impair recovery from voluntary eccentric exercise. Endurance exercise has been shown to alter EV profiles Oliveira et al. Chronic exercise in murine models 3weeks swim training was, for example, shown to significantly increase serum EV count Bei et al.

Both EV count and modal EV size were elevated in race horses following a single bout sustained km endurance exercise Oliveira et al. Alternatively, in humans Fruhbeis et al. However, the concentration of plasma-EVs remained elevated after exhaustive running.

In murine models, Oliveira et al. It is therefore of interest that in the current study we did observe a shift in EV modal size towards larger EVs, at 48h post-EIMD Supplementary Figure 1 , albeit this trend was not statistically significant. Our findings are also in line with previous work of Lovett et al.

Thus, it may be that exercise duration, intensity, and modality, in addition to differential species responses may yield variable results, and this warrants further exploration to fully understand effects of an acute exercise bout on circulating EVs.

Indeed, as already noted, great individual variability is observed in human responses to various exercise modalities, and thus differing EV profile response may in part underlie differing adaptation to these modalities Trovato et al.

Alternatively, in lieu of changes to the number and morphology of circulating EVs, their transported cargo may be more relevant to the exercise response, and thus future studies may choose to examine this variable. Unlike research on endurance models, the current literature is lacking in resistance training investigations, and specifically eccentric muscle-damaging protocols, such as those used in the current study.

Whilst Cui et al. Our study provides evidence that early changes in EV profile following EIMD significantly correlate with subsequent changes in CK, a known biomarker of muscle damage, and thus acute changes in EV profile post-exercise may indicate subsequent magnitude of muscle damage.

Both processes may result from the mechanical EIMD stimulus e. Alternatively, it is tempting to speculate that the EV response may be causative of subsequent changes in muscle damage markers, such as CK; however, such causality is not possible to ascribe with the data collected here.

Outside of EIMD, other types of muscle damage, such as laser membrane ablation and cellular hypoxia, have been reported to induce rapid increases in EV release; however, these have hitherto been performed on either zebrafish Middel et al.

Whilst associations have previously been observed between EV release profiles in response to inflammatory disorders Hosseinkhani et al. Therefore, we were interested in examining any putative age differences in EV release profiles between older and younger individuals following a bout of EIMD.

Whilst our results presented here suggest no major differences in EV modal size or EV plasma concentration in younger vs. older individuals, following a single bout of EIMD, some caution should be taken in the interpretation of these results due to the small sample size assessed and volunteer selection.

The findings presented here are with recreationally active younger and older participants, all participants habitually engaged in structured physical activity, and thus are not representative of wider physically inactive Western populations Farrell et al.

Importantly also, in the ageing population, reduced physical activity and increase in sedentary time are typically observed Lindsay et al. Furthermore, no difference in muscle mass or fat mass was seen in our study population, unlike that witnessed in wider society Volpi et al.

By studying highly active ageing cohorts, we can separate physiological differences of ageing from inactivity induced changes Harridge and Lazarus, Our results, therefore, should be interpreted in light of the relatively physical trained cohort presented here.

Any potential differences suggested by the results presented here between age groups may be enhanced when expanding this study to exercise naive younger and older individuals; however, this may reflect effects of long-term inactivity, not ageing per se.

Whilst this pilot study on EIMD has presented some interesting results in relation to EVs as putative biomarkers for muscle damage, these findings will need further validation in larger cohorts that can be guided in sample size collection by the results presented here.

Future investigations should also conduct in depth analysis of EV cargo composition will be of considerable interest for the identification of EV-related biomarkers in EIMD.

Therefore, it will be of great interest to perform full EV profiling analysis using RNA sequencing, proteomics and metabolomics to reveal the EV cargo profiles in response to EIMD, also in different age populations.

Whilst EV cargo biomarkers have been implicated in the pathophysiology of inflammation-associated disorders, research regarding their role in EIMD and ageing remains limited. This study therefore provides the first insights into the potential of EV-profiling in association with muscle-damaging exercise and ageing and paves the way for future studies, aiming to extend current knowledge on their roles as mediators of health-promoting effects, and as biomarkers, associated with physical activity.

In conclusion, here we show that physical responses to eccentric exercise induces plasma-EV changes that correlate with CK release post exercise, a biological marker of muscle damage.

EV profiles did not appear to change significantly in relation to age groups assessed active younger vs. older , which importantly may make them a reliable biomarker to assess effects of exercise interventions across age groups.

As EV release has previously been associated with small animal models of muscle damage, our study further supports that EV release profiles immediately following exercise may also play a role in the EIMD response in humans.

If the post-exercise EV response does indeed reflect physiological injury recovery responses, the magnitude and content of EV profile changes could be of interest for strategies to reduce the impairing effects of EIMD.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The studies involving human participants were reviewed and approved by College of Liberal of Arts and Sciences Research Ethics Committee, University of Westminster, United Kingdom.

YK and BE: conceptualisation, methodology, project administration, and writing — original draft preparation. YK, IK, and SL: formal analysis and investigation.

YK: human fitness testing. YK, IC, IK, SL, and BE: data curation. YK, IK, SL, and BE: visualisation. BE: supervising and funding acquisition. YK, IC, SL, and BE: writing — review and editing.

All authors contributed to the article and approved the submitted version. BE was supported by the Quintin Hogg Charitable Trust. This work was supported in part by publication funds provided by the British Society for Research on Ageing.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

The authors would like to acknowledge the time and dedication of all participants who participated in this study. The authors would also like to thank Evangeline Tanner for her assistance with data collection of human participants Dr Andrew Dalby for consultation on statistics, and the Guy Foundation for funding the purchase of equipment utilised in this work.

Supplementary Figure 1 Representative example of EV profiles responses of a younger and older participant, showing shifts of EV modal size to larger EVs in response to EIMD during the post-exercise recovery period.

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These pre-conditioning interventions produced no measurable change in indirect biomarkers of EIMD e. CK , and did not produce DOMS, inflammation or an extended reduction in force production [ 17 , 18 , 19 ]. Furthermore, participants exposed to various pre-conditioning activities demonstrated significantly lower CK and DOMS values, whilst force production measures recovered sooner after muscle-damaging exercises, when compared with control groups.

However, pre-conditioning strategies also have a short duration of effectiveness and are required to be performed 1 day to 2 weeks prior to any muscle-damaging activity, with 24—48 h as the preferred time period for isometric contractions [ 15 , 17 , 20 ] and weeks for non-damaging eccentric muscle contractions [ 21 , 22 , 23 ].

Whilst not fully understood, it is believed that pre-conditioning activities exhibit protection against EIMD using similar mechanisms to that of traditional RBE, such as neurological priming, extra-cellular matrix remodelling and pennation angle changes to provide a short-term adaptation, allowing greater transmission of force and improved joint congruency while maintaining neural excitation [ 4 , 24 , 25 , 26 ].

More recent evidence also indicates a smaller displacement of the myotendinous junction as a potential of the pre-conditioning effect on muscle damage [ 27 ]. Therefore, the pre-conditioning effect and RBE both exhibit protection from EIMD following the subsequent muscle-damaging bouts and are underpinned by similar mechanisms.

However, with the pre-conditioning effect, the exercises during the first bout are deliberately selected to lower the level of EIMD e. lower intensity, lower volume, shorter muscle length in preparation for a more intense second bout of muscle-damaging exercises.

Conversely, the RBE phenomenon is usually considered when the muscle-damaging exercises are prescribed similarly, if not identically, between the first and subsequent bouts with the intention to cause a high level of EIMD following the first bout.

Given the ability to induce an RBE with minimal EIMD symptomatology, pre-conditioning activities may be a useful tool for rehabilitation or concurrent training scenarios where high levels of EIMD are expected, although are not desirable.

For example, attenuating EIMD for athletes returning to resistance training after a long hiatus because of an injury may decrease the recovery time needed between rehabilitation sessions, and thereby speed up the return to play time [ 14 ]. Individuals considering introducing resistance exercises as part of their normal training programme, such as endurance athletes or youth athletes, may also consider pre-conditioning activities to minimise the level of EIMD during the initial period of a resistance training programme.

There is clear evidence to date regarding the effectiveness implementing pre-conditioning activities prior to muscle-damaging protocols to ameliorate the level of EIMD.

However, the methodologies used to implement pre-conditioning thus far have varied. For example, including short- and long-duration MVC appears to have a potent prophylactic effect against EIMD when used prior to high-intensity eccentric contractions [ 10 , 17 ].

Low-intensity eccentric contractions, low-volume eccentric contractions and downhill walking have been performed on various muscle groups, attenuating a second more intense bout of eccentric contractions without first causing EIMD [ 20 , 23 , 28 ].

Because of the variety of contraction times, intensities and volumes used for pre-conditioning all reducing EIMD with some degree of effectiveness, it is difficult for the practitioner to discern best pre-conditioning practice.

A recent review examining the effect of isometric pre-conditioning highlighted that isometric pre-conditioning attenuates EIMD in populations with little or no prior eccentric contraction exposure.

However, the review highlighted that pre-conditioning has not yet been explored in a trained population and may not be effective, owing to the long-term prophylactic effects of eccentric contractions [ 29 ].

This review also postulated that the potency of attenuation of EIMD may be related to the number of maximal isometric contractions. However, to date, no appraisal of the quality of studies examining isometric or EPC has been completed, nor a meta-analysis conducted based on pooled data of multiple studies.

Therefore, a systematic review and meta-analysis are warranted to provide insight into the effectiveness of these different pre-conditioning strategies. Consequently, the aim of the current systematic review and meta-analysis is to evaluate the effectiveness of pre-conditioning strategies in preventing EIMD, give some insight to practitioners regarding implementation and to highlight areas of further research.

This systematic review and meta-analysis used the PRISMA Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines [ 30 ] for the methodology and reporting of data Fig. Selection process detailing the search procedure for assessing eligibility for inclusion in a systematic review and meta-analysis.

Intervention studies with a parallel between-group design to examine the prevention and severity of muscle-damaging contractions following a bout of pre-condition exercises e. Comparison outcome measures were compared between groups based on measures at 24 h, 48 h, 72 h and 96 h after the muscle-damaging protocol.

Outcome outcome measures included common markers of indirect muscle damage e. CK, DOMS, joint range of motion [ROM] and muscular contractility measures e. isometric force and isokinetic torque.

Studies were excluded if: 1 they were reported in a language other than English with no translation; 2 they were clinical in nature i. participants were recovering from a trauma such as a stroke or injury ; 3 no clear measures of EIMD were reported; 4 no comparative groups were presented; and 5 they were reported as abstracts, reviews or case studies.

The following literature search was performed on 7 April, across three electronic databases PubMed, CINAHL and Scopus. An equivalent free text search was used for the Cinahl and Scopus databases.

Furthermore, reference lists from included studies were screened as a supplementary search. A list of abstracts extracted from the literature search was screened for eligibility by two authors with sport and exercise science backgrounds LB and BDT. Abstracts were classified as either meeting the inclusion criteria yes or not meeting the inclusion criteria no.

Once screening was completed, the full texts of all included abstracts were further screened using the same inclusion criteria. Any discrepancies for the screening of the abstracts and the full-text search between the two authors involved a third author KD , until a consensus was reached.

Upon completion of the abstract and full-text screening, information relating to the study design, number of participants, study aims and main findings was extracted. For studies where outcome measures were reported as figures, an e-mail requesting raw data was sent to corresponding authors.

When a response was not provided by the corresponding authors, data from figures were extracted using a digitising software ImageJ; National Institutes of Health, Bethesda, MD, USA. The methodological quality of each study was assessed using the Kmet appraisal checklist for quantitative studies [ 31 ].

Given that several of the Kmet criteria were inapplicable for the distinct study designs employed by the included studies, these criteria were either modified or replaced, with potential confounding variables common within the study design of EIMD studies. Criterion 5 if random allocation to a treatment group was possible, is it described?

was modified to the following: a score of 2 if participants were matched by MVC or another appropriate baseline measure and randomised with the description of the method used; a score of 1 if participants were matched by MVC or other criteria but not randomised, or a method of randomisation was not mentioned; a score of 0 if participant allocation to a group was not clearly described.

Criterion 6 interventional and blinding of investigators to intervention was possible, is it reported? was modified to the following: if baseline or pre-test measures included eccentric or isometric contractions, was a washout period observed prior to treatment commencing?

A score of 2 if adequate time was allocated to allow for the cessation of any RBE occurring from the contraction present in the study; a score of 1 if concentric contractions were measured only, some time may be present between testing and treatment; a score of 0 if eccentric or isometric contractions were present and time allocated between baseline or pre-test measures not adequate to extinguish a repeated bout effect.

Criterion 7 If interventional and blinding of subjects to intervention was possible is it reported? was modified to the following: were measures of dependent variables taken at the same time of day or within a specified window?

A score of 2 if the time of day that dependent variables were measured was specified in the methodology or elsewhere in the study ; a score of 1 if the time of day was not specified but a similar time of day could be assumed based on evidence elsewhere, for example, figures or tables; a score of 0 if the time of day was not specified in the methodology and cannot be assumed based on other information present in study.

Criteria Controlled for confounding? were modified to: were dependent variables recorded at appropriate intervals? A score of 2 if dependent variables were measured daily with no less than 2 consecutive days post-intervention; a score of 1 if dependent variables were measured at regular intervals but may skip some h periods; a score of 0 if dependent variables were measured randomly with no reasoning behind the schedule.

The certainty of evidence was also assessed using the Grading of Recommendations Assessment, Development and Evaluation GRADE. The certainty of evidence for each outcome was examined by two authors KD and US using the Grading of Recommendations Assessment, Development and Evaluation GRADE protocol with ratings that ranged from very low to high levels of certainty [ 33 , 34 , 35 ].

A meta-analysis was conducted to determine the effectiveness of pre-conditioning strategies for EIMD prevention using the RevMan version 5.

The outcome measures were compared between the control group no pre-conditioning and pre-conditioning group at 24 h, 48 h, 72 h and 96 h after the muscle-damaging protocol.

For studies where a control group was not included, the group that performed equivalent exercises between pre-conditioning i. first bout and the subsequent muscle-damaging protocol i. second bout was treated as the control group.

For example, in the study by Nosaka et al. first bout. All the participants then performed 24 maximal eccentric contractions 2 weeks later as their muscle-damaging protocol i. second bout. In this instance, we extracted data following the pre-conditioning activity i.

first bout for the 24ECC group and treated these data as our control group. The data extracted following the muscle-damaging exercises i. bout 2 from 2 and 6ECC were treated as the pre-conditioning groups in our meta-analysis. Once all data were extracted from each study, forest plots were generated to determine differences between groups using the random-effects model.

The magnitude of differences i. pre-conditioning vs control groups was calculated based on standardised mean differences, with values of 0. The between-group differences based on the pooled data were quantified as Z -values from the forest plot, in conjunction with p -values to determine the level of statistical significance, and the alpha level set at 0.

Thus, effectiveness of the pre-conditioning activities was ascertained according to standardised mean differences and statistical significance between the pre-conditioning and control groups. A total of abstracts were extracted and screened according to the inclusion criteria from PubMed, Scopus and CINAHL databases.

Following screening, abstracts were excluded, and the remaining 61 full-text articles were further assessed, leaving 23 articles for inclusion Fig. Primary outcome measures included in the current review were common measures associated with EIMD; biochemical markers, physical performance, post-exercise muscle soreness, swelling and degrees of joint motion.

These measures have previously been used as strong indicators of EIMD. Biochemical markers of indirect muscle damage included CK and myoglobin, whilst physical performance measures included MVC, maximal isometric torque, peak isometric force, peak eccentric force, peak concentric force and maximal eccentric contraction.

Post-exercise muscle soreness was classified based on a subjective rating of muscle soreness using visual analogue scales. Swelling was measured externally by measuring the girth circumference in millimetres of the affected limb segment.

Degrees of joint motion were taken as the percentage of change in degrees of the affected limb, or a true change in degrees, symptomatic of reduced contractility and tissue swelling. Studies that included all groups with pre-conditioning activities had a total sample size of participants Table 1.

The average age, height and body mass including range for isometric pre-conditioning and ECC pre-conditioning groups were Similar characteristics were present for studies with those that had a control group i. no pre-conditioning activity , for which the total sample size was participants with an average age, height and body mass including range of For the research design, 11 studies included a control group with no pre-conditioning activities, whilst eight studies incorporated groups where all participants performed pre-conditioning activities.

Various types of interventions were used as pre-conditioning strategies to prevent EIMD Table 2 , with the most common being low-intensity or low-volume eccentric contractions 11 studies , followed by maximal isometric contractions six studies , Smith machine squatting one study , and downhill or level walking one study.

One study used both isometric and eccentric contractions as pre-conditioning, whilst one study used maximal isometric and eccentric contractions in separate groups. A variety of exercises were used to cause EIMD Table 2 , with the most common being eccentric contractions 15 studies followed by downhill running two studies , downhill walking one study and squatting one study.

The most common biomarkers for indirect muscle damage were CK 18 and myoglobin One study did not measure blood biomarkers of EIMD. The level of DOMS was reported by 19 studies using visual analogue scales of 1—10, 1— or 1—, with greater ratings indicating higher perceived soreness.

Muscle swelling was reported using upper arm girth eight studies , thigh girth two studies and ultrasound six studies. Physical performance was measured using MVC of either the elbow flexors or knee extensors eight studies , MVC torque of either the elbow or knee extensors ten studies , running economy one study and 3-km time trial one study.

In addition, 11 studies examined ROM of the elbow joint or knee joint. Time between pre-conditioning and muscle damaging protocols varied among studies; some studies utilised more than one time period between groups. The most common durations between pre-conditioning and muscle-damaging protocols were 2 days eight studies , 2 weeks eight studies , 1 week six studies and 3 weeks five studies.

The Kmet values for included studies ranged from good to excellent Table 3. All included studies met the following criteria: eligibility criteria; similar measures at baseline and days 1—4; participants shared a similar training background; use of a pre-conditioning protocol; EIMD protocol performed more than 24 h after pre-conditioning; reported two or more outcome measures associated with EIMD e.

CK, myoglobin, ROM, MVC, DOMS or muscle girth ; and results reported with measures of central tendency and dispersion.

When examining individual studies that compared various pre-conditioning protocols, the contraction type, intensity, volume, and joint angle of pre-conditioning activities and the duration between pre-conditioning activities and the muscle-damaging protocol appeared to influence the magnitude of protection from EIMD following subsequent muscle-damaging exercises.

With respect to the volume of pre-conditioning activities, a greater volume of the pre-conditioning activity performed before the muscle-damaging exercises also exhibited lower levels of EIMD, such as the comparison between 10 repetitions of MVC to two repetitions of MVC held for 3 s during each MVC [ 10 ] and six eccentric contractions to two eccentric contractions [ 18 ].

However, results from one study showed no differences in the level of EIMD between pre-conditioning activities of 10 and 30 eccentric contractions [ 28 ]. Downhill walking for 5 min also exhibited lower levels of EIMD as a pre-conditioning activity when compared with flat walking for 5 min [ 16 ], further supporting greater protection from EIMD with a higher intensity of pre-conditioning exercises.

When comparing contraction types, eccentric contractions exhibited a greater protection from EIMD than isometric contractions [ 38 ]. Finally, pre-conditioning activities consisting of 30 repetitions of MVC held for 3 s during each contraction performed at an elbow angle of 20° lengthened elbow flexors showed greater protection from EIMD than at an elbow angle of 90° shortened elbow flexors.

When examining the acute responses to the pre-conditioning exercises, no significant differences in EIMD markers i. However, a significant increase in DOMS and a reduction in MVC were reported after 10 and 30 maximal eccentric contractions for at least 72 h post-exercise [ 28 ].

However, in the same study [ 38 ], DOMS was significantly increased, whilst MVC was decreased for only 24 h after 30 repetitions of MVC at 90° of an elbow flexion angle. Differences between baseline and post-exercise were not reported after the pre-conditioning activity for EIMD in one study [ 18 ], although CK and DOMS were notably increased for at least 48 h after 2 and 6 repetitions of maximal eccentric contractions, with 6 repetitions of maximal eccentric contractions exhibiting significantly greater EIMD than 2 maximal eccentric contractions.

The current systematic review and meta-analysis examined the extent to which pre-conditioning interventions attenuate EIMD. Applying stringent inclusion criteria, 19 articles were included in the meta-analysis. Based on meta-analytical data, pre-conditioning interventions attenuated the level of EIMD i.

CK, DOMS, ROM and MVC when performed a minimum of 24 h prior to a bout of strenuous exercise, compared with groups without pre-conditioning activities. In addition, the participants recovered from EIMD more quickly when exposed to pre-conditioning activities, with notably greater SMD values from 24 h post-exercise to 72 h post exercise.

Whilst several of the pre-conditioning activities employed in these studies also increased the level of EIMD elevated CK and DOMS and decreased MVC , the level of EIMD was notably lower than in the control group after the EIMD protocol.

Thus, implementing pre-conditioning activities as a priming method prior to the initial exposure of muscle-damaging exercises alleviates symptoms of EIMD, and may accelerate recovery. According to the traditional concept of RBE, a high level of EIMD is expected following the first bout of muscle-damaging exercises to minimise the level of EIMD following the second bout of similar, or identical muscle-damaging exercises [ 6 , 11 ].

However, studies in this review achieved a similar prophylactic response to the RBE using pre-conditioning, bypassing the need for an initial high level of EIMD response. Pre-conditioning activities may provide an alternative with the benefits of the RBE that can be tailored for a specific outcome.

A potent RBE brought about by traditional means of EIMD such as heavy strength training may attenuate subsequent muscle damage for months [ 2 , 13 ]. However, the duration of protection from the pre-conditioning intervention appears to be specific to the type of intervention chosen, with low-volume maximal isometric pre-conditioning IPC utilising between two and ten isometric contractions attenuating muscle damage for only a few days 2—4 days [ 10 , 15 , 17 ], while higher volume IPC appears to maintain attenuation potential for several weeks [ 13 , 19 ].

The use of eccentric pre-conditioning has also been shown to maintain protective capacity against EIMD for weeks to months using low-volume maximal eccentric contractions to attenuate a second bout of high-volume ECC of the same exercise [ 18 , 28 ].

The data in this review show that IPC interventions using between 2 and 60 isometric contractions provide adequate attenuation from CK, DOMS, ROM and MVC [ 10 , 15 , 19 ]. Several mechanisms have been proposed to explain how pre-conditioning activities attenuate muscle damage in the absence of any noticeable stimulus.

Type IV collagen within the extracellular matrix ECM may interact with isometric and low-intensity ECC, allowing for a slow contraction of the type IV fibres.

The alignment of ECM in this way may improve the transduction of force through the musculoskeletal system [ 43 ]. The slow contraction of the ECM may explain why EIMD will occur when exercise is completed immediately after pre-conditioning, although attenuated 24 h later.

Furthermore, eccentric contractions as pre-conditioning activities have been suggested to upregulate the cytoskeletal proteins, which may strengthen the cytoskeletal protein network by stabilising the sarcomeres during eccentric contractions in subsequent muscle-damaging bouts [ 44 ]. It has also been suggested that pre-conditioning exercises may upregulate anti-oxidant enzymes whilst concomitantly reducing inflammatory markers, which may attenuate the secondary muscle damage response following subsequent muscle-damaging bouts [ 45 , 46 ].

The duration of protection, particularly from maximal isometric contractions, is short lived, lasting only several days. It is therefore possible that non-damaging contractions may prevent EIMD by facilitating greater force output and muscle fibre recruitment as a result of familiarisation.

In addition, muscle length appears to be an important factor in the success of a pre-conditioning strategy, as force production is greatly affected by muscle length afforded by the angle of a joint [ 47 , 48 , 49 ]. In the study by Chen et al. Ofori et al.

This allowed for greater fibre recruitment while taking advantage of the mechanical and neural properties afforded by fibre elongation. The current meta-analysis exhibited significantly lower levels of EIMD for the pre-conditioning group from various interventions, suggesting that non-damaging protection against EIMD from subsequent strenuous training sessions is achievable using a variety of pre-conditioning strategies.

However, the mechanisms offering protection are still debatable, and require more exploration beyond the scope of this review. Several studies included in this review used sub-maximal isometric or concentric MVCs during familiarisation and baseline testing to reduce interference with the chosen pre-conditioning strategy [ 10 , 13 , 37 ].

While it is generally accepted that concentric contractions do not confer muscle damage at low volumes [ 1 , 50 ], it remains unclear as to what extent concentric contractions affect the outcomes associated with EIMD.

Similarly, the use of sub-maximal isometric contractions during familiarisation may have conferred some degree of pre-conditioning. Whilst this was not evident in the data within this review, the exploration of isometric thresholds that exhibit protection from EIMD may be valuable in broadening the application of pre-conditioning.

Research implementing MVC and ECC as outcome measures should also consider how the implementation of MVC at baseline will impact their outcomes. The studies included in this review highlight that pre-conditioning can affect MVC, ROM and other measures of performance for up to 3 weeks, while some studies have shown that a single bout of ECC can have a RBE lasting several months [ 18 ].

This review corroborates suggestions made by Chen et al. It is unclear exactly which pre-conditioning strategies would be effective for use prior to athletic performance. However, based on data collected from this review, it is possible to speculate that pre-conditioning may attenuate EIMD resulting from strenuous training.

Similarly, pre-conditioning may improve concurrent training outcomes. Pre-conditioning could be implemented into periodisation plans to reduce EIMD between training sessions, improving the quality of cardiovascular training that occurs post-resistance training, particularly for endurance athletes for whom resistance training is less frequent or when commencing resistance training after a break or as part of a new training regime.

This would ultimately lead to greater training quality, allowing full ROM and force production while reducing the injury risk during training for endurance athletes [ 16 , 20 ].

The complex nature of sporting competition makes speculation regarding the effectiveness and appropriate application of pre-conditioning for athletes challenging. Further evaluation is therefore required to explore the use and effectiveness of pre-conditioning in sport.

Many studies in this review utilised mono-articular pre-conditioning interventions. Mono-articular movement does not replicate the complexity of most training exercises, utilising only a single joint and only the muscles that cross that joint.

Multi-articular pre-conditioning offers a greater opportunity for pre-conditioning to transfer to the real world, using multiple joints and muscles working at varying angles across an entire limb [ 19 , 20 , 22 ].

Lima et al. The EIMD induced by downhill running was attenuated by the isometric leg press as measured by CK, MVC and muscle soreness; however, running kinematics and metabolism were not significantly different between groups.

This suggests the prophylactic effects of pre-conditioning can be transferred across different exercises if the same muscle groups are targeted. No studies included in this review implemented multiple exercises targeting the same muscle group in their second bout designed to cause EIMD.

Traditional resistance training sessions typically consist of multiple exercises with a focus on developing a particular athletic attribute power, strength, strength endurance. Huang et al. It therefore remains unclear to what extent EIMD will be attenuated by a multi-articular pre-conditioning activity following a bout of traditional resistance exercises that are performed with multi-articular activities.

During methodological quality assessment of studies in the current systematic review, several areas were identified as generally scoring low using the Kmet criteria. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

The authors would like to acknowledge the time and dedication of all participants who participated in this study. The authors would also like to thank Evangeline Tanner for her assistance with data collection of human participants Dr Andrew Dalby for consultation on statistics, and the Guy Foundation for funding the purchase of equipment utilised in this work.

Supplementary Figure 1 Representative example of EV profiles responses of a younger and older participant, showing shifts of EV modal size to larger EVs in response to EIMD during the post-exercise recovery period.

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Corresponding author. Keywords: Eccentric , resistance training , repeated bout effect. JOURNAL FREE ACCESS. Published: December 03, Received: August 13, Available on J-STAGE: May 04, Accepted: December 03, Advance online publication: - Revised: -. Download PDF K Download citation RIS compatible with EndNote, Reference Manager, ProCite, RefWorks.

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