It is well known that unaccustomed exercise, particularly lengthening contractions, results in muscle damage (18). In the days after lengthening contractions, extensive disruption of the ultrastructure of skeletal muscle occurs, muscle proteins (e.g., creatine kinase (CK)) are released into the blood, and delayed onset of muscle soreness (DOMS) appears (18). These indices of muscle damage are substantially reduced when the same muscle-damaging exercise is performed several weeks later, indicating a rapid adaptation of skeletal muscle to lengthening contractions (14). This phenomenon has been referred to as the repeated bout effect (18). Although several mechanisms have been proposed to explain the repeated bout effect, the way skeletal muscle adapts to lengthening contractions remains largely unknown (18).
Numerous human studies have investigated the effects of non-muscle-damaging exercise on oxidative stress of tissues (mainly blood and skeletal muscle), but only a few have addressed the effects of acute muscle-damaging exercise in humans (5-7,11,17). The most interesting finding reported by these studies is that disturbances in some indices of blood oxidative stress may persist for and/or appear several days after muscle-damaging exercise, which is in direct contrast to the return to the resting values a few hours after an acute non-muscle-damaging exercise (2,4,6,24). Given that secondary muscle damage after muscle-damaging exercise is mainly induced by phagocyte infiltration into muscle (3,25), and that infiltrating phagocytes generate reactive species (8,19), it is probable that a part of the oxidative stress originates from reactive species produced by phagocytes after lengthening contractions. In fact, it has been suggested that reactive species contribute to secondary muscle damage after lengthening contractions (22,29).
An important preliminary step in ascertaining the possible biological function of reactive species in lengthening contraction-induced muscle damage is the determination of the temporal relationship between blood indices of oxidative stress and indices of muscle damage. The adaptation to lengthening contractions provides an interesting model to study this relationship, because responses to a damaging exercise (bout 1) can be compared with the responses to a relatively nondamaging exercise (bout 2).
The primary purpose of the present study was to examine the effect of muscle-damaging exercise on the time-course changes in several indices of muscle function and damage (i.e., maximal isometric torque, range of motion (ROM), DOMS, and blood CK activity) and compare them with changes in blood oxidative stress indices, to explore potential relationships among these phenomena. Secondly, we sought to determine whether a bout of muscle-damaging exercise could influence the response of markers of muscle damage and blood oxidative stress after a second bout of muscle-damaging exercise performed approximately 3 wk later. We hypothesized that muscle-damaging exercise would increase oxidative stress that would be sustainable for days after exercise, and that this increase would be attenuated after a second exercise bout.
MATERIALS AND METHODS
Subjects.
Twelve healthy females (age 23 ± 2 yr, height 165 ± 2 cm, weight 54 ± 3 kg, and body fat 24.9 ± 2.0%) who were exercising for less than 3 h·wk−1 participated in this study. Subjects had no experience with muscle-damaging exercise training for at least 6 months before the study and were not taking any medications, oral contraceptives, or dietary supplements. They were instructed to abstain from strenuous exercise for 7 d before and during data collection. All volunteers were eumenorrheic (reporting their menstrual cycle as lasting 24-30 d). The muscle-damaging exercise trial fell within the luteal phase of the menstrual cycle (2 up to 6 d after ovulation), in an attempt to avoid any variance in estrogen levels between the first and second exercise bouts. A written informed consent to participate in the study was provided by all participants after the volunteers were informed of all risks, discomforts, and benefits involved in the study. The procedures were in accordance with the Helsinki declaration of 1975, and approval was received from the institutional review board.
Design.
Volunteers performed two isokinetic lengthening contraction bouts separated by 24-30 d, depending on the duration of their menstrual cycle. In each of the two exercise sessions, volunteers had to accomplish five sets of 15 lengthening maximal voluntary isokinetic contractions at an angular velocity of 1.05 rad·s−1 in the prone position. A 2-min rest interval was incorporated between sets. The exercise protocols were undertaken by all participants in their dominant leg (hamstrings), whereas the other leg served as a control. All physiological and biochemical measurements were determined before, immediately after, and 1, 2, 3, 4, and 7 d after exercise. These measurements were assessed after both lengthening contraction bouts. All measurements and blood samplings were performed between 9:00 and 11:00 a.m. after an overnight fast and having abstained from alcohol and caffeine for 24 h.
Muscle damage indices.
An isokinetic dynamometer was also used for the measurement of the isometric knee flexors' peak torque at 90° knee flexion. The best of the three maximal voluntary contractions was recorded. The test-retest reliability of isometric peak torque measurement was 0.98. The assessment of pain-free ROM was performed manually. The investigator moved the calf at a very low angular velocity from 90° knee flexion to the position where the subject felt any discomfort. The test-retest reliability of ROM measurement was 0.96. Each subject assessed DOMS by palpation of the muscle belly and the distal region of the hamstrings in a seated position with the muscles relaxed. The assessment of soreness of the exercised lower limb was also performed during walking. The test-retest reliability of DOMS by palpation and by walking measurement were 0.94 and 0.92, respectively.
Blood collection and handling.
Directly after taking the blood sample, 1 mL of 5% trichloroacetic acid (TCA) was added to 1 mL of whole blood collected in EDTA tubes. The samples were centrifuged and the clear supernatants were collected. Another portion of blood was collected in plain tubes, left on ice for 20 min to clot, and centrifuged at 1500g for 10 min at 4°C for serum separation. Serum was used for the determination of CK, thiobarbituric acid-reactive substances (TBARS), protein carbonyls, catalase, uric acid, bilirubin, and total antioxidant capacity (TAC). Blood samples were stored in multiple aliquots at −30°C and were thawed only once before analysis.
Assays.
GSH was assayed according to Reddy et al. (23,27). Twenty microliters of whole blood treated with TCA were mixed with 660 μL of 67 mM sodium potassium phosphate (pH 8.0) and 330 μL of 1 mM 5,5′-dithiobis-2-nitrobenzoate (DTNB). The samples were incubated in the dark at room temperature for 45 min, and the absorbance was read at 412 nm.
GSSG was assayed according to Tietze (27). Two hundred sixty microliters of whole blood treated with TCA were neutralized up to pH 7.0-7.5 with NaOH. Four microliters of 2-vinyl pyridine were added, and the samples were incubated for 2 h at room temperature. Five microliters of whole blood treated with TCA were mixed with 600 μL of 143 mM sodium phosphate (6.3 mM EDTA, pH 7.5), 100 μL of 3 mM NADPH, 100 μL of 10 mM DTNB, and 194 μL of distilled water. The samples were incubated for 10 min at room temperature. After addition of 1 μL of glutathione reductase, the change in absorbance at 412 nm was read for 3 min.
TBARS was assayed according to Keles et al. (16). One hundred microliters of serum were mixed with 500 μL of TCA 35% and 500 μL of Tris-HCl (200 mM, pH 7.4) and incubated for 10 min at room temperature. One microliter of 2 M Na2SO4 and 55 mM thiobarbituric acid solution were added, and the samples were incubated at 95°C for 45 min. The samples were cooled on ice for 5 min and were vortexed after adding 1 mL of TCA 70%. Finally, the samples were centrifuged at 15,000g for 3 min, and the absorbance of the supernatant was read at 530 nm.
Protein carbonyls were assayed according to Patsoukis et al. (21). In 50 μL of serum, 50 μL of 20% TCA was added, incubated in the ice bath for 15 min, and centrifuged at 15,000g for 5 min at 4°C. The supernatant was discarded, and 500 μL of 10 mM 2,4-dinitrophenylhydrazine (in 2.5 N HCL) for the sample, or 500 μL of 2.5 N HCL for the blank, were added in the pellet. The samples were incubated in the dark at room temperature for 1 h, with intermittent vortexing every 15 min, and were centrifuged at 15,000g for 5 min at 4°C. The supernatant was discarded, and 1 mL of 10% TCA was added, vortexed, and centrifuged at 15,000g for 5 min at 4°C. The supernatant was discarded, and 1 mL of ethanol-ethyl acetate (1:1 v/v) was added, vortexed, and centrifuged at 15,000g for 5 min at 4°C. The washing step was repeated two more times. The supernatant was discarded, and 1 mL of 5 M urea (pH 2.3) was added, vortexed, and incubated at 37°C for 15 min. The samples were centrifuged at 15,000g for 3 min at 4°C, and the absorbance was read at 375 nm.
Catalase activity was assayed according to Aebi (1). In 20 μL of serum, 2975 μL of 67 mM sodium potassium phosphate (pH 7.4) was added, and the samples were incubated at 37°C for 10 min. Five microliters of 30% hydrogen peroxide were added to the samples, and the change in absorbance was immediately read at 240 nm for 1.5 min.
TAC was assayed according to Janaszewska and Bartosz (15). In 20 μL of serum, 480 μL of 10 mM sodium potassium phosphate (pH 7.4) and 500 μL of 0.1 mM 2,2-diphenyl-1 picrylhydrazyl were added and incubated in the dark for 30 min at room temperature. The samples were centrifuged for 3 min at 20,000g, and the absorbance was read at 520 nm.
Total protein in serum was assayed using a Bradford reagent. CK was assayed using an enzymatic method based on the rate of NADPH formation that absorbs at 340 nm (13), with a kit from Spinreact (Sant Esteve, Spain). Uric acid was assayed using a kit from Assel (Rome, Italy) on the basis of formation of a red-colored compound that absorbs at 510 nm (9). Bilirubin was assayed using a kit from Assel on the basis of formation of a red-colored azobilirubin that absorbs at 550 nm (26).
Each assay was determined spectrophotometrically in triplicates (duplicates for CK) on the same day to eliminate variation in assay conditions and within 1 month of the blood collection. Estradiol was determined in duplicates. The intraassay coefficient of variation for each measurement was estradiol 2.9%, CK 3.9%, GSH 4.1%, GSSG 6.1%, TBARS 4.7%, protein carbonyls 5.4%, catalase 7.0%, uric acid 3.4%, bilirubin 2.9%, and TAC 3.4%.
Dietary analysis.
Participants were asked to record their diet for 3 d before the first exercise protocol and to repeat this diet before the second exercise protocol. Diet records were analyzed using the nutritional analysis system Science Fit Diet 200A (Sciencefit, Greece).
Calculations and statistical analysis.
The incremental or decremental area under the curve (AUC) for the 7-d protocol was calculated using the trapezoidal rule and by subtracting the area attributable to the baseline concentration. Effect size was calculated as the difference between bout 1 and bout 2 AUC concentrations divided by the pooled SD. Effect sizes of 0.2, 0.6, 1.2, 2.0, and 4.0 were considered to be small, moderate, large, very large, and nearly perfect, respectively, using a modified Cohen scale (http://newstats.org). The distribution of all dependent variables was examined by Shapiro-Wilk test and was found not to differ significantly from normal, except for CK values. Two-way ANOVA (bout × time) with repeated measurements on both factors were used to analyze isometric peak torque, DOMS, ROM, and all oxidative stress indices. CK activity was analyzed nonparametrically by Friedman's test. If a significant interaction was obtained, pairwise comparisons were performed through simple contrasts and simple main-effects analysis. Differences between the first and the second bouts with respect to incremental or decremental AUC were examined by Student's paired t-test. Correlation analysis between indices of muscle damage and indices of oxidative stress was done by Pearson's product-moment correlation. The test-retest reliability of the functional muscle damage indices was determined by performing the intraclass reliability test.
RESULTS
There were no significant differences in daily energy, macronutrient, and antioxidant intake between the first and second exercise protocols (P > 0.05; Table 1). Muscle damage indices are presented in Figure 1, and oxidative stress indices are shown in Figures 2-4. Incremental or decremental AUC of both muscle damage and oxidative stress indices are presented in Table 2.
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TABLE 1: Analysis of daily energy intake before bouts 1 and 2 of eccentric exercise (mean ± SD).
FIGURE 1: Isometric peak torque (A), range of movement (B), delayed-onset muscle soreness after palpation (C), and delayed-onset muscle soreness after walking (D) in the control (triangles) and experimental leg after bout 1 (open rectangles) and bout 2 (closed rectangles). Creatine kinase activity in the blood (E) after bout 1 (open rectangles) and bout 2 (closed rectangles) (mean ± SEM). * Significantly different from the preexercise value in the same bout (P < 0.05); # significantly different between bout 1 and bout 2 at the same time point (P < 0.05).
FIGURE 2: Glutathione (A), oxidized glutathione (B), and glutathione/oxidized glutathione (C) concentrations after bout 1 (open rectangles) and bout 2 (closed rectangles) (mean ± SEM). * Significantly different from the preexercise value in the same bout (P < 0.05); # significantly different between bout 1 and bout 2 at the same time point (P < 0.05).
FIGURE 3: Thiobarbituric acid-reactive substances (A) and protein carbonyl (B) concentration after bout 1 (open rectangles) and bout 2 (closed rectangles) (mean ± SEM). * Significantly different from the preexercise value in the same bout (P < 0.05); # significantly different between bout 1 and bout 2 at the same time point (P < 0.05).
FIGURE 4: Catalase activity (A) and uric acid (B), bilirubin (C), and total antioxidant capacity (D) concentrations after bout 1 (open rectangles) and bout 2 (closed rectangles) (mean ± SEM). * Significantly different from the preexercise value in the same bout (P < 0.05); # significantly different between bout 1 and bout 2 at the same time point (P < 0.05).
TABLE 2: Incremental or decremental area under the curve (AUC) per day in muscle damage and oxidative stress indices after bouts 1 and 2 of eccentric exercise (mean ± SD).
Muscle damage indices.
The main effects of bout and time, as well as their interaction, were significant with regard to isometric peak torque of the exercised leg (P < 0.001, P < 0.001, and P = 0.012, respectively; Fig. 1A). The value of isometric peak torque of the control leg was not significantly different between bouts 1 and 2. Therefore, only the values of the control leg after bout 1 are presented for clarity. Compared with baseline values, isometric torque of the exercised leg declined significantly up to 4 d after the first bout and returned to baseline values at 7 d. After the second bout, the decline in isometric torque was significant only up to 1 d after exercise and did not differ significantly from the resting value thereafter. Isometric torque of the control leg did not change significantly at any time point after either bout (P > 0.05).
Concerning ROM of the exercised leg, the main effect of bout and time, and their interaction, were significant (P < 0.001, P < 0.001, and P = 0.029, respectively; Fig. 1B). The value of ROM of the control leg was not significantly different between bouts 1 and 2. Therefore, only the values of the control leg are presented, for clarity. ROM of the exercised leg, compared with resting values, was decreased up to 3 d after the first bout and returned to baseline values at 7 d. After the second bout, the decrease in ROM lasted up to 2 d after exercise and did not differ significantly from the resting value thereafter. ROM of the control leg did not change significantly at any time point after either bout (P > 0.05).
The interaction of protocol and time, and both main effects, were significant with regard to DOMS after palpation (P = 0.011, P < 0.001, and P < 0.001, respectively; Fig. 1C) and after walking of the exercised leg (P = 0.031, P < 0.001, and P < 0.001, respectively; Fig. 1D). Compared with baseline values, DOMS after palpation and after walking of the exercised leg was increased up to 4 d after the first bout and returned to preexercise values at 7 d. After the second bout, the increases in both types of DOMS were, on average, lower and lasted for only up to 3 d after exercise. DOMS of both types of the control leg did not change at any time point after either bout (P > 0.05).
The main effects of bout and time, as well as their interaction, were significant with regard to serum CK activity (P < 0.001, P < 0.001, and P = 0.012, respectively; Fig. 1E). Compared with resting activity, CK was increased at 3 and 4 d after the first exercise bout, whereas after the second bout the increase in CK was, on average, lower and was significant only at 4 d after exercise.
Glutathione status.
The main effects of bout and time, as well as their interaction, were significant with regard to GSH (P = 0.044, P = 0.022, and P = 0.034, respectively; Fig. 2A), GSSG (P = 0.004, P = 0.030, and P = 0.024, respectively; Fig. 2B) and GSH/GSSG (P = 0.016, P < 0.001, and P = 0.041, respectively; Fig. 2C). Exercise decreased GSH, increased GSSG (not after the second bout), and decreased GSH/GSSG at several time points after both exercise bouts. However, the number of significant differences through time after the first bout was much higher than for those after the second bout (8 vs 3).
TBARS and protein carbonyls.
The interaction of protocol and time, as well as both main effects, were significant with regard to TBARS concentration (all at P < 0.001; Fig. 3A). Bout 1 increased TBARS at 2, 3, and 4 d after exercise, whereas bout 2 did not affect TBARS concentration (P > 0.05). With regard to protein carbonyls, the main effects of bout and time, as well as their interaction, were significant (P < 0.001, P < 0.001, and P = 0.017, respectively; Fig. 3B). Protein carbonyl concentration was higher at 2, 3, and 4 d after bout 1 compared with the resting value, whereas its concentration after bout 2 was not different at any sampling point (P > 0.05).
Catalase, uric acid, bilirubin, and TAC.
Regarding catalase activity, the interaction of protocol and time, as well as both main effects, were significant (P = 0.008, P = 0.021, and P < 0.001, respectively; Fig. 4A). Catalase activity was higher at 2, 3, and 4 d after bout 1 compared with the resting value, whereas its concentration was not different after bout 2 at any sampling point (P > 0.05). Concerning uric acid, the interaction of protocol and time, as well as both main effects, were significant (P = 0.012, P = 0.032, and P < 0.001, respectively; Fig. 4B). Uric acid concentration was higher at 2 and 3 d after bout 1 compared with the resting value, whereas its concentration after bout 2 was different only at day 3. Regarding bilirubin, the interaction of protocol and time, as well as both main effects, were significant (P = 0.002, P < 0.001, and P < 0.001, respectively; Fig. 4C). Bilirubin concentration was higher at 2, 3, and 4 d after bout 1 compared with the resting value, whereas its concentration after bout 2 was different only at day 3. The main effects of bout and time, as well as their interaction, were significant with regard to TAC (P = 0.044, P = 0.029, and P = 0.048, respectively; Fig. 4D). Compared with resting values, TAC increased at 3 d after the first exercise bout, whereas after the second bout the increase in TAC was, on average, lower and significant only at 3 d after exercise.
Correlation among indices of muscle damage and oxidative stress.
Pearson's test revealed many significant correlations for almost any combination between postexercise changes in muscle damage and oxidative stress indices. However, no strong significant correlation coefficients (> 0.70) were evident between postexercise changes in any of the variables examined (range 0.13-0.70, median value of 0.26 for all postexercise time points). No single muscle damage or oxidative stress index proved optimal for assessing the extent of oxidative stress or muscle damage, respectively. Additionally, no single time point in the postexercise period proved optimal for assessing the extent of muscle damage, oxidative stress, or both. The only moderate exception was the correlation between changes of isometric torque with changes in oxidative stress indices after exercise. This index of muscle damage exhibited the highest correlation coefficients with changes in the oxidative stress indices compared with any other index of muscle damage (i.e., DOMS, ROM, CK) independent of the postexercise time point examined. In these correlation pairs (i.e., loss of isometric torque and oxidative stress indices), the highest correlation coefficients appeared between the loss of isometric torque immediately after exercise, with changes in oxidative stress indices at 3 d after exercise after bout 1; only these are presented for the sake of completeness (Fig. 5A-I).
FIGURE 5: Correlation between percent decrease in isometric torque immediately after exercise and percent change in oxidative stress indices at 3 d after exercise compared with preexercise values. All correlations were significant (P < 0.05).
DISCUSSION
To our knowledge, this is the first investigation on the effect of repeated muscle-damaging exercise on the blood (or any other tissue) oxidative stress of animal or humans. The present results reveal that lengthening contractions uniformly (i.e., in a similar pattern through time) modified the levels of the selected oxidative stress indices, indicating increased oxidative stress in the blood, peaking in all indices except for bilirubin at 3 d after exercise and returning toward baseline afterwards. As predicted by the repeated bout effect phenomenon, the indirect indices of muscle damage changed dramatically after bout 1, but much less after bout 2. Accordingly, the magnitude of the circulating oxidative stress responses was much higher after the first bout of exercise compared with that induced by the same bout performed 3-4 wk later.
Calculation of incremental or decremental AUC for analysis of serial postexercise measurements.
In this study, our interest lay not only in the way muscle damage and oxidative stress indices changed with time but also in the total response of these indices. To get this information, incremental or decremental AUC was calculated. This summary measure gives a more honest indication of the magnitude of change of these indices (with data that have been collected through the whole observation period) and increases the chances of locating a significant difference between the first and second bouts. Additionally, from a biological perspective, it gives a fairly good picture of the concentration changed "sensed" by a human organism during a study period, because it is reasonable that the impact of the changes in oxidative stress biomarkers on muscle function and metabolism will depend on both the magnitude and the duration of these changes.
Blood oxidative stress during recovery from muscle-damaging exercise.
Oxidative stress can be estimated by determination of reactive species-induced damage in lipids, proteins, and other molecules, as well as by measurement of activity and/or concentration of antioxidant molecules (10). Because of the complexity of redox homeostasis, a battery of measurements have been suggested to reliably monitor changes in oxidative stress (12). TBARS and protein carbonyls are widely used markers of lipid and protein oxidation, respectively (12). One of the main enzymatic antioxidant compounds is catalase, and GSH is one of the main nonenzymatic compounds (10). TAC represents the ability of serum to neutralize reactive species and is greatly affected by changes in concentration of uric acid and bilirubin (10).
There were large disturbances in the levels of all oxidative stress and antioxidant molecules measured after bout 1 and much less after bout 2, as indicated by the effect sizes calculated through incremental or decremental AUC values. The effect sizes calculated produced values ranging from large (−1.8 in GSH and GSH/GSSG) to nearly perfect (+6.8 in protein carbonyls); the median in absolute value was equal to 3.6 (considered very large). In sum, these findings provide evidence that oxidative stress was highly decreased after a repeated bout of muscle-damaging exercise, and this adaptation seems to be a secondary response to a reduced degree of exercise-induced muscle damage.
The increase in TAC after exercise suggests that acute muscle-damaging exercise activates the antioxidant defenses of the body, a case that was also evident immediately after acute non-muscle-damaging exercise in a previous study from our laboratory (20). Indeed, the postexercise kinetics of uric acid and bilirubin (two major antioxidants of the blood) resembled those of TAC, indicating a relationship between these two antioxidant molecules with TAC. Additionally, the increased activity of catalase after lengthening contractions is consistent with the increased TAC. Nevertheless, this increase in antioxidants did not prove efficient at inhibiting the increase in glutathione, lipid, and protein oxidation of the blood.
An alternative explanation for the increased oxidative stress after muscle-damaging exercise may be a decreased clearance rate of these biomarkers in the days after exercise. Regarding glutathione status, for example, it is probable that after muscle-damaging exercise there might be an increase in the blood clearance of GSH (e.g., increased consumption in muscle), which would also explain a decrease in blood GSH concentration. Direct testing of the possible involvement of clearance rate in the effect of muscle-damaging exercise on oxidative stress is warranted.
Correlation analysis.
Changes in isometric torque exhibited the highest correlation coefficients with changes in the oxidative stress indices compared with any other noninvasive index of muscle damage. Given that decreases in maximal muscle torque is the best index of muscle damage (28) and that muscle damage induces and/or is induced by oxidative stress (25), it is reasonable to assume that the larger the magnitude of decrease in muscle strength, the greater the changes of oxidative stress biomarkers will be. Indeed, the highest correlation coefficients appeared between the loss of isometric torque immediately after exercise with changes in oxidative stress indices at 3 d after exercise after bout 1 (i.e., where the highest changes in both muscle damage and oxidative stress indices were observed). It is worth noting, however, that the amount of common variation between any two variables (expressed as the square of the correlation coefficient between two variables) produces coefficient of determination values (r2) of low (7% in catalase) to moderate (49% in protein carbonyls) magnitude. The magnitude of these values suggests a low relationship between muscle damage and oxidative stress.
Origin of blood oxidative stress after muscle-damaging exercise.
Although no direct evidence of muscle damage can be provided, the large attenuation in isometric torque, ROM, DOMS, and CK values after bout 2 provides indirect evidence that differences in muscle damage existed after the two lengthening contraction bouts. Subsequently, the large attenuation in all blood oxidative stress indices after bout 2 can be largely attributed to decreased muscle damage after bout 2 compared with bout 1.
After lengthening contractions, neutrophils and other phagocytic cells are activated and recruited to the site of the initial damage (10). Those immunity cells produce O2− with the catalytic action of the reduced nicotinamide-adenine dinucleotide phosphate (NADPH)-oxidase system (10). During O2− production, two O2 molecules are needed, so this reaction is called the oxidative burst. O2− can be converted to H2O2 by superoxide dismutase (10). H2O2 is not a free radical, but it is considered a reactive species because of its toxicity and capacity to cause reactive species formation. In leukocytes, myeloperoxidase transforms H2O2 in HOCl−, one of the strongest physiological oxidants (10). These reactants, if they stay unchecked, can also destroy adjacent healthy muscle tissue. As Close et al. (8) have commented, this oxidative burst has been directly shown to occur approximately 3 d after lengthening contractions in a mouse model and indirectly in humans after downhill running. Consequently, the major part of the delayed oxidative stress production that follows muscle-damaging exercise probably comes from neutrophils and macrophages that are recruited to the site of the initial damage. That said, it is conceivable that the repeated bout effect may occur, in part, because of a decrease in oxidative stress and subsequent attenuation of the lengthening contraction-induced muscle damage, and/or the repeated bout may induce less muscle damage, less invasion of leukocytes, and subsequent attenuation of oxidative stress.
Methodological applications.
The present work indicates that measuring the levels of blood oxidative stress parameters immediately and/or for some hours after muscle-damaging exercise produces results of limited value, because for 4 d after this type of exercise, the levels of these parameters may remain altered. Therefore, future relevant studies should perform multiple blood samplings late into recovery (at least more than 4 d and less than 7 d, because at this time point, all indices had returned to resting levels) to describe the effects of exercise in more complete dimensions. Additionally, researchers designing experiments in the field of oxidative stress should take care to have all participants abstain from muscle-damaging exercise for more than 4 d before enrollment in the study, to ensure the "resting" condition of the blood oxidative stress.
CONCLUSIONS
To our knowledge, this is the first report on the repeated bout effect from the side of oxidative stress. The first main finding is that lengthening contractions provoked large, uniform alterations in several oxidative stress indices and antioxidant molecules in women, all of them indicating increased oxidative stress. The second main finding is that a repeated bout of lengthening contractions induced much less muscle damage and blood oxidative stress than the first bout. It is suggested that muscle-damaging exercise should be viewed as a different challenge compared with non-muscle-damaging exercise with regard to their effects on blood oxidative stress.
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