Eccentric exercise effect on blood oxidative-stress markers and delayed onset of muscle soreness : Medicine & Science in Sports & Exercise

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Eccentric exercise effect on blood oxidative-stress markers and delayed onset of muscle soreness


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Medicine & Science in Sports & Exercise: March 2002 - Volume 34 - Issue 3 - p 443-448
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Muscle soreness is common in activities involving eccentrically biased muscle actions (3,4,5,10,14,19). Delayed onset of muscle soreness (DOMS) typically occurs 8–10 h after exercise (5,16) and peaks between 24 and 72 h after exercise (10,16). Creatine kinase (CK), a marker of muscle damage, has been shown to increase in the blood after eccentric exercise (5,6,10,14–16,19,28). Elevated activity of this enzyme, which typically does not leak out of undamaged cells, has been used as an indication of muscle membrane damage or changes in permeability of the plasma membrane.

Exercise-induced oxidative stress and reactive oxygen species have been suggested to be contributory mechanisms of muscle damage (4,5,14,23,27,29). The alteration in blood flow and the infiltration of inflammatory factors such as neutrophils and monocytes have been suggested to contribute to secondary muscle damage (4,14,22,25,29). Plasma and muscle cytokines, such as IL-2 and IL-6, were reported to be increased after eccentric exercise that resulted in muscle damage (7,9). It is unclear whether exercise-induced damage by eccentric contractions would demonstrate alterations in oxidative-stress markers in blood. Saxton et al. (29) reported that muscle malondialdehyde (MDA) and plasma thiobarbituric acid-reactive substances (TBARS) were not significantly altered after serial repetitions of maximal voluntary eccentric exercise. Conversely, McBride et al. (23) reported a positive relationship between high-intensity resistance exercise and plasma MDA. However, both MDA and TBARS are either indirect or insensitive markers of free-radical–mediated processes. Recently, Alessio et al. (1) reported that repeated isometric exercise at 50% of maximal voluntary force increased lipid hydroperoxides immediately after and 1 h post exercise. In contrast, MDA and protein carbonyls (PC) were not significantly altered in the blood.

The ratio of reduced (GSH) to oxidized (GSSG) glutathione in the blood has been used as a sensitive index of exercise-induced free radical production to aerobic exercise (9,14,30,33). Only one study has examined whether GSSG/GSH has a relationship to DOMS and CK response after eccentric contraction (8). No significant change in blood glutathione was reported in response to the eccentric exercise (EE). The eccentric exercise protocol was downhill running rather than high-tension eccentric contractions, which might have a different influence on blood glutathione status.

The oxidation of amino acids to form PC has been demonstrated to occur in rat muscle in response to exercise (20,27). Reznick et al. (27) reported that PC were increased after a single bout of exhaustive EE in rat muscle. The formation of PC appears to be related to muscle production of an intermediate resembling the hydroxyl radical (20). Oxidized proteins also have increased probability of proteolytic degradation and were shown to increase in urine in rats in a similar manner to the oxidized proteins of skeletal muscle (20). Therefore, PCs seem to be a fairly stable product of oxidative stress, whereas glutathione status is dependent on the reduction status, which can be rapidly modified. The appearance of plasma PC in response to high-intensity EE has not been evaluated in humans. Therefore, the purposes of the present investigation were to examine whether GSSG/TGSH ratio and PC were altered in blood after a single bout of 60 EEs and whether there was a significant relationship of DOMS and CK response to these markers of free-radical production.



Eight healthy men (18–32 yr) volunteered to participate as subjects after all procedures were explained, a medical history questionnaire completed, and informed consent signed. The University Human Subjects Committee approved all experimental procedures. The medical history questionnaire screened potential volunteers for any health-related problems that might affect the parameters measured. All subjects did not drink alcohol on a regular basis and had not actively engaged in resistance exercise for 6 months before the study. Subjects were instructed to maintain their normal diet during the study and brought in daily food records each day of the study. Subjects did not take antiinflammatory drugs or nutritional supplements throughout the study.

Measurements of height, weight, and percent body fat by three skinfold sites were obtained to characterize the subjects (18). Within 7 d, subjects returned to the laboratory to perform the exercise protocol. Initial measurements for range of motion (ROM), and both flexed (FA) and relaxed arm angle (RA) of the elbow in both the dominant and nondominant arms were obtained using a goniometer (12). The angles were determined a minimum of three times on both arms. If the angle was >2° different among the determinations additional measurements were conducted. The average of the three measured angles (within 2°) was used for both FA and RA. The ROM was calculated as the difference between the FA and RA positions.

Muscle soreness (DOMS) values were obtained using a visual analog score (VAS 10 cm long) (11). The scale ranged from no muscle soreness (value of 1) to very, very sore muscle soreness (value of 10). Subjects were instructed to place a line from the left side (no muscle soreness) toward the amount of soreness they perceived in the relaxed and palpated situation. Subjects also indicated on a picture of the arms the location of their soreness. Subjects indicated their soreness for both dominant and nondominant arms. The soreness score was calculated as the distance in cm from the left end of the scale.

Blood samples (17 mL) were drawn from an antecubital vein by using sterile techniques. The maximum isometric force (MIF) was then obtained for both dominant and nondominant arms. The MIF was also used to ascertain the weight needed for the EE protocol. Each subject was seated on a biceps preacher-curl machine and the center of the elbow joint placed on a pad. Subjects were strapped into the curl machine and their chest placed against the pad of the machine. A strain gauge (model no. SM-250–38, Interface Inc. Scottsdale, AZ) was attached to the machine and linked to an IBM-compatible computer. The force was analyzed using a software program from National Instruments (Austin, TX) and was calibrated with known weights. The MIF was recorded with the subject’s elbow joint angle at 90° at a sample rate of 1000 Hz. The subject performed three maximum contractions first with the dominant arm and then after resetting the subject’s position with their nondominant arm. Subjects were allowed a period of 30-s rest between trials.

Measurement order was maintained throughout the entire experiment. Within 1 h after initial measurements, each subject performed a single bout of EE and then the measurements were again obtained. Subjects were instructed to return to the exercise physiology laboratory at 24, 48, 72, and 96 h after the EE. All blood samples were immediately processed and then either the plasma or treated samples stored in a −90°C freezer until analyzed.

Eccentric exercise.

Subjects performed the eccentric contractions by using their nondominant arm. Subjects were seated into the machine, and their nondominant arm was placed on the arm-curl pad into the proper position (90°). The amount of weight lowered was based on the formula, (1.35 × MIF of the dominant arm) + 2.43 kg. The initial load on the nondominant arm approximated 150% of their MIF. The subjects were instructed to lower the weight to an arm angle of 170° in 3 s. Two spotters then lifted the weight within 7 s. The subjects were encouraged to lower the weight 60 times during the 10 min. If the subject was unable to lower the weight in a controlled manner in the 3 s, a portion of the weight (1.1 kg) was removed. All subjects performed 60 eccentric contractions, and all had the weight decreased by 2.2–4.4 kg.

Blood collection and preparation.

Blood was collected from the antecubital region by Vacutainer using sterile techniques by a trained phlebotomist. Samples were obtained from the dominant arm before the EE and from the nondominant arm after EE (at 10 min). Approximately 17 mL was obtained and immediately placed on ice and processed for glutathione status (10 mL). The rest of the blood was centrifuged at 3000 rpm for 15 min at 4°C in a Beckman (J2-21) centrifuge (Beckman, Fullerton, CA) to obtain plasma. The plasma was pipetted into microcentrifuge tubes and stored in a −90°C freezer until analyzed for CK and PC. Plasma CK was determined by using Sigma Kit 520 (St. Louis, MO). All samples were determined in duplicate and compared to known standards on a Shimadzu UV-1601 spectrophotometer (Shimadzu, Columbia, MD) at 520-nm wavelength.

Whole blood was used for determination of glutathione status. Total blood glutathione (TGSH) and oxidized glutathione (GSSG) were analyzed using 5,5′-diothiobis-2 nitrobenzoic acid (DTNB) to combine with GSH to form 5-thio-2-nitrobenzoic acid (TNB). GSSG was reduced back to GSH by glutathione reductase in the presence of NADPH. The rate of TNB formation was determined at 412 nm and was proportional to the sum of GSH and GSSG (2).

Blood samples mixed with EDTA were treated with two volumes of ice-cold 10% 5-suflosalicylic acid containing bathophenantrolinedisulfonic acid (BPDS) concentration of 1 mM for TGSH. BDPS was used to chelate metal ions. This mixture was centrifuged at 10,000 ×g for 15 min at 4°C in a Beckman (J2-21) centrifuge. The supernatants were used for determination of TGSH. The determination of GSSG used whole blood (2 mL) with EDTA and was treated immediately with ice-cold 10% 5 sulfosalicylic acid (1 mL) containing BDPS (1 mM). The solution was mixed and then centrifuged at 10,000 ×g at 4°C in a Beckman (J2–21) centrifuge for 15 min. The supernatant was neutralized with NaOH (pH 7.0–7.5), and 2 μL of 2-vinylpyridine was added per 100 μL of supernatant and vigorously mixed for 5 min to derivatize GSH as recommended (17). The resultant supernatants were then analyzed in duplicate.

Protein carbonyls were determined from plasma by using the dinitrophenolhydrazine (DNPH) spectrophotometric method (32). Plasma protein was determined by the Lowry method (21) and then adjusted to 4 mg·mL−1 protein with phosphate buffer. Samples were run through columns containing Sephadex G 10 and rinsed with 2N HCl. The effluent was collected and the absorbance determined at 360 nm. All samples were measured in duplicate and compared the change in absorbance with and without DNPH. Values are expressed as molar quantities using the extinction coefficient, 22,000 M−1·cm−1 as previously reported (32). The coefficient of variability of resting samples was < 8%.

Statistical analyses.

The data were statistically analyzed using a repeated measures 1 × 6 ANOVA. When a significant time effect was obtained, a Tukey’s post hoc test was used to identify where the differences were. Pearson product moment correlations were determined comparing the DOMS and CK responses to both the glutathione and PC data to ascertain whether there were relationships between these variables. Statistical significance was set at a P ≤ 0.05. The data are presented as means ± SE.


The characteristics of the subjects are presented in Table 1. The subjects were of average height and weight and had normal amounts of body fat for young college males. The amount of force for the dominant arm was about 9% greater than the nondominant arm before the EE. All subjects successfully completed 60 eccentric contractions.

Table 1:
Subject characteristics.

The effect of the EE on muscle function parameters and DOMS is presented in Table 2. The comparison of the dominant arm and nondominant arm over time on each of the variables is presented. All subjects maintained their MIF for the dominant arm over the times measured. In contrast, a significant (P ≤ 0.0001) decrease force production as indicated by MIF in the nondominant occurred immediately after the EE. The decline in MIF was significant (P ≤ 0.0002) at the times measured (Table 2).

Table 2:
Muscle function and DOMS in response to eccentric exercise.

There were no changes in RA, FA, or range of motion (ROM) in the dominant arm throughout the times measured (Table 2). Significant differences were noted in both RA (P < 0.001) and FA (P < 0.0005) in the nondominant arm after EE. The decrease in RA occurred immediately after the EE and was maintained below normal throughout the study. There was an increase in FA immediately after EE, and this was above normal through 48 h after the EE. Range of motion (RA–FA) was significantly decreased after the EE and remained at this level through 72 h after EE.

There were no significant changes in the amount of muscle soreness (DOMS) in the dominant arm over time. In contrast, the nondominant arm had significant muscle soreness from 24–96 h after EE (Table 2). The highest soreness value recorded was at 48 h and then returned toward normal levels by 96 h.

Creatine kinase was significant elevated in the blood after the EE (P < 0.0005) (Fig. 1). The amount of CK in the blood increased significantly at 48 h peaked at 72 h and then decreased slightly by 96 h. Compared with the resting value, there was an eightfold increase in CK at 72 h after the EE.

Plasma creatine kinase response to eccentric exercise;N = 8 for each time point; ★ significant difference compared with rest value;P ≤ 0.05.

Glutathione status in whole blood as indicated by GSH, GSSG, and TGSH did not significantly change over time after the EE. The ratio of GSSG/TGSH is presented in Figure 2. Total glutathione (TGSH) remained between 0.73 and 0.87 mM at all times measured. GSSG was 0.17 mM before the EE and remained between 0.16 and 0.20 mM at all times determined. GSH was 0.54 mM before the EE and demonstrated a tendency for a slight but insignificant decline from 24 to 96 h after EE. The ratio of GSSG/TGSH was 19% before EE and was not altered immediately after the EE (20.4%). This ratio tended to increase to 24% at 72 and 96 h after the EE but did not reach significance.

Blood GSSG/TGSH ratio in response to eccentric exercise;N = 8 for each time point; ★ significant difference compared to rest value;P ≤ 0.05.

Plasma PC concentration immediately after the EE was not significantly different than resting values (Fig. 3). However, PC concentration was significantly elevated (P ≤ 0.008) at 24 (83%) and 48 (62%) h after the EE. By 72 h, the PC concentration had returned to normal levels.

Plasma protein carbonyls in response to eccentric exercise;N = 8 for each time point; ★ significant difference compared to rest value;P ≤ 0.05.

Relationship of DOMS and CK to GSSG/TGSH and to PC.

There was no significant relationship between DOMS and CK to GSSG/TGSH. In contrast, DOMS scores were significantly correlated to the PC concentration over time. The Pearson product moment correlation indicated an r = 0.498 (P ≤ 0.0003). In contrast, CK and PC values did not demonstrate a significant correlation over time (P = 0.10).


The present investigation is the first study to report that an EE protocol, which resulted in DOMS, elevated blood CK, and decreased ROM and MIF, can also result in elevated plasma PC levels in humans. In addition, a significant relationship between DOMS and plasma PC in the blood was noted. This study also reports that whole-blood glutathione status was not significantly changed at any of the time points measured. These results taken together suggest that PC probably is a more suitable oxidative-stress marker for examining muscle damage to eccentric actions. This may be related to the fact that PCs are more stable whereas glutathione changes are reversible.

Eccentric contractions can manifest DOMS and elevated blood CK levels (6,13,16,23,24). The possible mechanisms that may contribute to skeletal muscle damage include free-radical–mediated processes and inflammatory factors within the muscle and the musculotendinous junction (4,7,9,14,22). The present investigation was designed to determine whether protein carbonyls and/or blood glutathione status, which are sensitive markers of oxidative stress, would be altered in response to a high-intensity EE. The results from this investigation substantiate that DOMS and blood CK increase dramatically after a single bout of EE with a decrement in muscle function and range of motion. This study is the first to report that plasma PC concentration will increase 24–48 h after the EE. Furthermore, the results indicate that PC appears to be a better marker of oxidative muscle damage compared with whole-blood glutathione status.

Limited information is available concerning the response of muscle to EE and radical species production. Data on the glutathione status as an indicator of free radical production in blood after EE are lacking. Furthermore, comparison of DOMS and plasma CK activity with PC concentration and blood glutathione status has not been investigated. Few studies have examined oxidative stress in response to EE and muscle soreness (8,23,27,29). The results are inconsistent, perhaps in part due to the mode of exercise employed and the markers of oxidative stress. Resistance exercise was noted to increase MDA in the blood although only at a high intensity (23). In contrast, downhill running (29) and whole-body resistance exercise to failure (Boyer, personal communication) did not significantly alter blood MDA despite demonstrating muscle soreness.

Glutathione status in the blood has been previously reported to change transiently as a result of aerobic exercise of sufficient intensity and duration (9,33) but not downhill running (8). Typically, a decrease in the amount of GSH and an increase in GSSG indicate oxidative stress has occurred. The ratio of GSSG/TGSH has also been used because total glutathione levels may change in the blood (7). In the present investigation, the amount of oxidized glutathione was similar to that which was reported for downhill running (8). The glutathione status was unchanged by EE, downhill running (8), or lowering weights as in the present investigation. This suggests that, based on blood glutathione status, there was little if any oxidative stress in the blood in response to the EE. This is in agreement with the reports with MDA (29).

It is possible that the glutathione status in the present study was transiently altered as a result of the EE but was not indicated because we did not obtain the blood sample until 15–20 min after the EE. This could have given sufficient time for the GSSG to be converted back to GSH. However, our results suggest that there is a tendency for slightly greater amounts of oxidized glutathione to the total glutathione from 24–96 h after the EE. This nonsignificant trend needs further investigation since only eight subjects were examined and muscle glutathione status was not examined.

Another possibility is that glutathione status changed in the muscle but was not in the blood. Additionally, there could have been a transient change in the blood glutathione status within the 4–24 h after the EE when leukocyte infiltration might be at its height (14,24,31). In contrast, PC content in plasma was significantly increased at 24 and 48 h after EE, suggesting that oxidative stress occurred in response to the EE. It is important to note that PC levels did not increase immediately but were only significantly elevated at 24 and 48 h after the EE.

Saxton et al. (29) reported that PC content in leg muscle was significantly increased after concentric leg exercise rather than EE. In their study, PC increases in the muscle was attributed to the metabolic stress that occurred during the concentric bout of exercise. It is unclear whether the elevated plasma PC observed in the present study originated from the muscles and or from other tissues. However, it was recently noted that urinary PC can be quantified nicely and demonstrate a parallel change as muscle PC (20). It was also reported that high-intensity anaerobic running to exhaustion increased PC derivatives in rat lung (26). Additionally, isometric exercise to failure was recently shown to increase lipid hydroperoxides in blood immediately and 1 h after exercise (1). This study also reported that PC in blood only increased in response to aerobic exercise. However, no time point past 1 h after the exercise was examined. Finally, it is interesting to note that the increase in PC in the present investigation significantly correlated to the increase in DOMS. However, the increase in PC coincided with increases in DOMS for the 24- and 48-h time frame only although DOMS was elevated for 72 h after the EE.

In conclusion, the present study is the first investigation to report that protein carbonyls in blood increase in response to EE, which resulted in muscle soreness, decreased ROM and muscle force, and elevated blood CK activity. Blood glutathione status was not altered over the times measured. Furthermore, the changes in plasma PC significantly correlated with the changes in DOMS. These results suggest that oxidative stress does occur in response to high-intensity EE, and this occurs within the first 48 h after EE.

Address for correspondence: Allan H. Goldfarb, Ph.D., Department of Exercise and Sport Science, University of North Carolina Greensboro, Greensboro, NC 27402-6169; E-mail: [email protected]


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