Each repetition of MVC led to a trough and a peak in [O2Hb] and [HHb] recordings. The troughs and peaks were detected and the difference between the baseline value (averaged over 0.5 seconds epoch baseline before the peaks and troughs) and the corresponding value at the troughs and peaks were calculated. The differences were averaged across the repetitions of MVC and termed as [ΔO2Hb] and [ΔHHb]. A sum of [ΔO2Hb] and [ΔHHb] was also calculated to reflect the changes in total hemoglobin ([ΔtHb]). The [ΔtHb] is considered as an indirect measure of changes in the local muscle blood flow (14). A percentage of the difference between [ΔtHb] at instants after the bouts of ECC compared with the value before ECCs was calculated for the 7 NIRS channels. A grand percentage was calculated as an average ± SEM of the percentage difference of [ΔtHb] at all the channels. Maps of local muscle blood flow for the TA muscle were generated using the percentage difference of [ΔtHb] over the 7 channels. The interpolation was performed using an inverse distance weighted interpolation to obtain an image of the [ΔtHb] distribution map (3).
Surface EMG was collected from the TA muscle using a bipolar surface electrode configuration (Neuroline 72001k; Ambu A/S, Ballerup, Denmark). The electrodes were placed on the muscle belly along the direction of the muscle fibers (interelectrodes distance: 2 cm). A single EMG channel was recorded as similar level of activity are reported in proximal and distal muscle portion of TA (7). The reference electrode was placed on the head of the tibia. Surface EMG was amplified (between 500 and 2000 times), bandpass filtered (5–500 Hz), digitized (12 bits A/D converter), and sampled at 2 kHz. For each session of isometric MVC, RMS values were estimated over 250-millisecond epochs moving in steps of 100 milliseconds. The calculated RMS values were averaged across all epochs. Similar to RMS values, MDF was estimated over 500-millisecond nonoverlapping epochs for each session of isometric MVC. The power spectrum for MDF estimation was computed using the Welch method by applying a 512 points Hanning window with 15% overlap between successive subepochs (40).
The normality of the entire data set was checked and confirmed by Shapiro-Wilk tests. Changes in average dependent variables, MVC force, mean RMS, mean MDF, [ΔtHb], pain/soreness scores, and pain area were compared using a linear mixed model (LMM) in SPSS (IBM SPSS Statistics version 19; IBM Corp., Armonk, NY, USA). Instants (before vs. immediately after vs. the day after), and sessions (ECC1 vs. ECC2 vs. control 1 vs. control 2) were introduced as within-subject factors of the LMM. An additional within-subject factor of channel (channels 1–7) was also added for [ΔtHb] recordings. To account for repeated measurements of the experiment, a repeated factor associated with the instants, sessions, and channels was added to the model. In control sessions, instead of 3 instants we had 2 instants (before vs. day after), but LMM is capable of handling such unbalanced data sets (41).
Bonferroni correction for multiple comparisons was used for post hoc test. The outcome measures in the control part of the experiment were analyzed for between-day reliability using relative (intraclass correlation coefficient [ICC]) and absolute estimates (SEM, minimum detectable change [MDC]). The measurements of the control sessions over 2 consecutive days (between days), and 2 days with 7 days in-between (between weeks) from the same 16 subjects were used for ICC, SEM, and MDC assessments. SEM was calculated using the following formula (Table 1):
where SD is the grand SD of the scores across all subjects (39). SEM is an indication of expected measurement error in a single individual's score. Minimum detectable change was calculated at the 95% level using the formula:
(39). SEM and MDC were reported using the same units as the mean values of the [ΔtHb], MVC, EMG RMS, and EMG MDF. In all tests, p ≤ 0.05 was considered statistically significant. Mean (SD) are reported. A grand average of [ΔtHb] was measured by making an average of [ΔtHb] over 7 channels. A linear regression (Pearson correlation coefficient) was used to test the correlation between grand [ΔtHb] and MVC force or mean RMS or mean MDF.
The between-day and between-week ICC, SEM, and MDC are reported in Table 1.
A statistically significant interaction between instant and session was detected for the isometric MVC force (p < 0.001; Table 2). Maximum voluntary contraction force decreased significantly immediately after both ECC1 and ECC2 (p < 0.001 and <0.01, respectively). The MVC force was significantly decreased the day after ECC1 (p < 0.001); however, it did not change significantly the day after ECC2. The isometric MVC force level immediately and the day after ECC2 was significantly higher compared with the same time points after ECC1 (p < 0.001 and <0.01, respectively).
Soreness Intensity and Soreness Area
A statistically significant interaction between instant and session was detected for the soreness intensity (p < 0.001). In the ECC1 session, the soreness intensity increased immediately after the exercise (p < 0.001) and the day after ECC (p < 0.001) compared with before ECC. In the ECC2 session, however, there was no significant difference between the soreness at any instants (before vs. after vs. the day after). The soreness intensity was significantly lower after the ECC2 (both immediately and the day after ECC2) compared with immediately and the day after ECC1. In both sessions, there was no significant difference between the soreness area at different instants (Table 2 and Figure 4).
For the total hemoglobin concentration [ΔtHb], a statistically significant interaction between instant and session was detected (p < 0.001; Table 2 and Figure 2). There was no difference between [ΔtHb] at baseline measures before ECC1 and at control days. The [ΔtHb] was significantly lower before ECC2 compared with before ECC1 (p < 0.01). In both ECC sessions, [ΔtHb] significantly decreased immediately after ECC compared with before ECC (p ≤ 0.05); and then it increased the day after ECC compared with immediately after ECC (p < 0.01). In the ECC1 session, the [ΔtHb] was still significantly decreased the day after ECC compared with before ECC (p < 0.001). However, there was no difference between the levels of [ΔtHb] before and the day after ECC2. The percentage difference of [ΔtHb] was significantly higher at immediately and the day after ECC1 than at immediately and the day after ECC2 (p < 0.001 and <0.001, respectively; Figure 2). Near-infrared spectroscopy channel played a significant role on [ΔtHb] (p ≤ 0.05). Generally higher [ΔtHb] was measured over the medial part of the TA (channels 3 and 6) compared with the lateral, proximal, and distal parts. However, this difference was consistent across all measurements (p ≤ 0.05; Figure 2).
In both ECC sessions, there was no significant difference between the mean EMG RMS at any instant (before vs. after vs. the day after). Median power frequency was similar at baseline measurement before ECC1 and at control days. A statistically significant interaction between instant and session was detected for MDF (p ≤ 0.05). Median power frequency decreased significantly at instants of ECC2 session compared with the same instants of ECC1 (p < 0.01; Table 2).
There was a positive correlation between MVC force and the grand [ΔtHb] during MVC (r = 0.59, p < 0.01); a negative correlation between EMG RMS and the grand [ΔtHb] during MVC (r = −0.21, p < 0.01); and finally a positive correlation between MDF and the grand [ΔtHb] during MVC (r = 0.23, p ≤ 0.05).
As hypothesized, the unaccustomed high-intensity ECC led to a significant decrease in [ΔtHb] at the day after ECC1, whereas repeating the same ECC did not. There was no difference between the MDF at control sessions and ECC1 session. Median power frequency was significantly lower at ECC2 session compared with ECC1. On the contrary, no changes were seen in RMS at any instants of the study. The data support the hypothesis that the local muscle blood flow and EMG spectral contents change with ECC experience. In other words, the local muscle blood flow and EMG spectral contents are altered after an unaccustomed ECC inducing muscle soreness. These alterations could provide a potential mechanism for a rapid adaptation that may decrease susceptibility of the muscle to develop further soreness in the subsequent ECC bout.
Principally, the ECC protocol was successful at inducing muscle soreness to TA (Figure 4). A reduction of 40% in muscle strength is regarded as one of the most valid and reliable indicators of muscle damage in humans (38). The protocol used in the present study induced a decline of 75% in muscle strength (17). It has been shown that such ECC protocol can produce DOMS, and reduction in range of motion for several days after the exercise (17,30). Hubal et al. (18) demonstrated that using a similar number of ECC contractions, large differences in muscle strength deficits were seen between participants after ECC exercise. Therefore, it is likely that different degrees of muscle damage and DOMS between different participants is an important limitation across DOMS studies. This limitation can be diminished using a protocol similar to the present study.
The relevance of NIRS measurements to monitor local muscle blood flow have been established previously (34); however, it should be noted that continuous wave NIRS using 2 wavelengths (850 and 760 nm) really does not reflect the absolute [ΔtHb] and it is just an indirect surrogate number that correlates with local muscle blood flow (13). The percentage difference of [ΔtHb] immediately after ECC2 and the subsequent day was significantly lower than observed at the same time points after ECC1 (Figure 2). This pattern resembles the RBE observed for outcome measures focusing on changes in pain sensitivity (pressure pain thresholds and nociceptive withdrawal reflex threshold [NWRT]) (17,19). Moreover, the [ΔtHb] before ECC2 was significantly lower than it was before ECC1, which could signal that ECC2 was performed while full recovery after ECC1 was not present. This is in line with the signs of central sensitization depicted by lower NWRT after ECC1 (17). Hosseinzadeh et al. (17) reported a decrease in NWRT after the strong stimulus induced by ECC1, whereas this was not observed after ECC2. The observed modulation of the NWRT indicates hyperexcitability of the spinal nociceptive pathways as a result of ECC1-induced muscle damage. This central facilitation in response to ECC1 provided an opportunity for rapid functional plasticity that allows to accommodate with ECC2 more efficiently, hence suggesting the importance of centrally mediated changes in relation to RBE (17). Furthermore, the lack of a significant decrease of [ΔtHb] the day after ECC2 may indicate that RBE can be detected by NIRS independent of full recovery after ECC1. Detection of RBE before full recovery substantiates further the role of neural adaptation mechanism in RBE (25,26).
In agreement with previous studies, MDF during MVC was significantly lower after consecutive bout of ECC, whereas RMS was unchanged (4,37). In parallel, MVC force decreased at the day after initial bout of ECC, whereas it returned to baseline the day after ECC2 (17). This can explain the lack of changes in RMS giving credit to a hypothesis suggesting an altered recruitment after ECC1 (4). Warren et al. (37) suggested that stable RMS amplitude after the initial and the consecutive ECCs reflects similar total neural activation between 2 ECCs. Chen (4) has suggested that the lower mean power frequency during ECC at 70% MVC reflects changes in muscle fiber activation pattern associated with RBE in biceps brachii muscle. The present study reproduced similar observations in TA during maximal isometric contraction and links the findings to the blood flow. Warren et al. (37) have suggested that the decrease in MDF can be due to a shift between the recruitment of faster motor unit to the activation of slower motor units. It should, however, be noted that the use of a single bipolar surface EMG channel does not enable the extraction of neural strategies identifying the physiological mechanisms responsible for the neural control of movement (10,11). Finally, 1 could suspect that the observed changes in MDF may be due to altered frequency modulation of the EMG activity through the conducting volume because of tissue swelling, etc. Hence, an increased attenuation of the high-frequency content of the EMG signals is realistic (as the result of DOMS). However, the attenuation of high-frequency content of the EMG signal would result in a decreased signal energy that should have been detected in the EMG RMS. Because we did not observe a significant change in EMG RMS, we believe that the observed changes in MDF cannot merely be explained by altered frequency response of the conducting volume.
Changes in intramuscular pressure, local blood supply, and motor unit recruitment pattern will all have an impact on [ΔtHb] during exercise (31). The change in the [ΔtHb], MVC force, and MDF could reflect a selective damage of fast-twitch glycolytic fibers after doing unaccustomed ECC1 (22). This may lead to recruitment of fewer fast-twitch fibers compensated by additional slow-twitch fibers for the repeated bout (ECC2) (37). Slow-twitch fibers have a higher oxidative capacity potential that contributes to the tissue oxygenation during consecutive ECC bouts compared with the initial bout of ECC. Better maintenance of the oxidative energy metabolism during consecutive ECC bouts could result in less muscle damage.
Another interesting observation in the present study was that ECC1 led to significant higher soreness compared with ECC2 (Figure 4 and Table 2). The pain distribution report before and after both bouts of ECCs can be addressed as evidence for attainment of TA muscle damage and soreness after ECC1 (Figure 4). A theoretical model of the relationship between muscle pain and central motor drive was proposed by Mastaglia (24). Under certain circumstances, contraction-induced pain during exercise may reduce the motor drive to the active muscles and thereby reduce the level of force production and the ability to sustain it. The significant decrease of the MVC at the day after initial bout of ECC in the existence of significant DOMS and moreover no DOMS at the day after ECC2 support this hypothesis (Table 2). Sadamoto et al. (1983) showed that blood flow to the biceps brachii muscle was completely impeded during sustained isometric contractions at 50% MVC, because of increased intramuscular pressure (32). The [ΔtHb] in our study was measured at 100% MVC. Although [ΔtHb] does not evaluate strictly the increase and decrease of blood volume, it gives an indication of the relative change in oxygen delivery and consumption by muscle blood volume (20). Thus, during a constant muscle work at 100% MVC, the changes in muscular force can impact [ΔtHb]. Therefore, in this condition, [ΔtHb] seems to be correlated with the changes in muscle force (see Results section regarding correlations) and a marker of muscle performance (the more force production the higher [ΔtHb]). Demura and Nakada (9) also reported such a relationship. Given the data related to the muscle soreness, MVC force, and [ΔtHb], one could infer that the increased soreness after ECC1 led to a decreased central motor drive resulting in a deficit in force production. Then, this decrease in force led to less [ΔtHb]. Finally, getting ECC experience and rapid potential adaptation to ECC1 mitigated the amounts of soreness in the subsequent ECC that can be seen as altered local muscle blood flow pattern and frequency contents of surface EMG after ECC2. Although the set level of exerted muscle force was the same across ECC bouts, the absolute intensity of 2 ECCs might have differed. As muscle cannot produce as high a force as the initial bout while it is not fully recovered, one might anticipate that the lower extent of DOMS after the second bout of ECC could be due to lower level of exerted muscle force. However, this kind of limitation is common among most of the studies investigating RBE (28,37). Furthermore, as explained in the above, choosing the same number of sets, repetition, and the same intensity of ECC aimed at minimizing this effect.
The local muscle blood flow and spectral contents of surface EMG changed following the first bout of ECC in line with our hypothesis. These changes were accompanied with signs of reduced muscle damage during the subsequent bout of ECC. The present study reproduced similar EMG activity findings but in the TA muscle and during maximal isometric contraction bridging these findings to changes in the local muscle blood flow. The relationship between the concomitant altered pattern of local muscle blood flow and frequency contents of surface EMG accompanied by the change in soreness after the unaccustomed ECC can be interpreted as a central adaptation mechanism to compensate for the peripheral changes in muscle capacity. The present study showed that preconditioning exercises consisting of high-intensity ECCs can produce protection against developing more muscle damage during subsequent bout of ECC. Therefore, clinicians, coaches, and strength and conditioning professionals are advised to emphasize preseason conditioning involving sports-specific movement drills of ECC to induce the protective and potentially analgesic effect of ECC. Further studies on RBE targeting other body regions are necessary to investigate the underpinning mechanisms of the protective effect.
The authors thank Mahdieh Hosseinzadeh for assistance with preparation of the Figure 3. The results of the present study do not constitute endorsement of the product by the authors or the National Strength and Conditioning Association. No funding was received for this work from any of the following organizations: National Institutes of Health (NIH), Wellcome Trust, Howard Hughes Medical Institute (HHMI), and other(s). The authors declare that they have no conflict of interest.
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Keywords:Copyright © 2015 by the National Strength & Conditioning Association.
local muscle blood flow; repeated bout effect; central adaptation; delayed onset muscle soreness