Effect of maximal-intensity exercise on systemic nitro-oxidative stress in men and women

Abstract

Objectives: The aim of this study was to test the hypotheses: (1) there is a negative correlation between protein and lipid oxidative damage following maximal-intensity exercise, and oxygen uptake and work intensity (%VO_2max) at the respiratory compensation point (RCP) in women and men; (2) nitro-oxidative stress following maximal-intensity exercise results from the intensification of anaerobic processes and muscle fibre micro-damage.

Methods: Study participants comprised 20 women (21.34±1.57 years) and 20 men (21.97±1.41 years) who performed a treadmill incremental test (IT); VO_2max: 45.08 ± 0.91 and 57.38 ± 1.22 mL kg⁻¹ min⁻¹ for women and men, respectively. The oxidized low-density lipoprotein (ox-LDL), 3-nitrotyrosine (3-NT) concentration and creatine kinase (CK) as well as lactate dehydrogenase (LDH) activity were measured in the blood serum, and total antioxidative capacity (TAC) and lactate concentration (Lac) were determined in blood plasma before and after IT.

Results: After the IT, increases in ox-LDL, 3-NT, CK, and LDH were seen in both groups (P < 0.05). After the IT, an increase in the TAC was only observed in women (P < 0.05). The post-exercise-induced increase in Lac was significantly higher in men than in women. Only in the group of women was a positive correlation (P < 0.05) between the post-exercise increase in TAC and changes in CK activity and LDH found.

Conclusions: The gain of ox-LDL and 3-NT following maximal-intensity exercise is independent of VO_2max, oxygen consumption and exercise intensity at RCP. This increase of ox-LDL and 3-NT is indicative of similar lipid and protein damage in women and men. A significant increase in TAC in women following maximal-intensity exercise is the result of muscle fibre micro-injuries.

Introduction

Maintaining prooxidative-antioxidant balance between reactive oxygen (ROS) and reactive nitrogen (RNS) species and antioxidant capacity is necessary for physiological intra- and intercellular signalling.^1,2 The hydroxyl radical (HO^•) and peroxynitrite (OONO⁻) arising in excess during muscle work (as a result of the severity of superoxide anion and nitric oxide synthesis) can cause damage to proteins and fats through oxidation and nitrosylation.^3,4 The result of this damage is an increase in oxidized low-density lipoprotein concentration (ox-LDL) and 3-nitrotyrosine (3-NT) in the blood.^4,5 The mechanism of ROS and RNS formation is dependent on the energy economy of the physical exercise.⁶ Some studies point to a positive correlation between maximal oxygen uptake (VO_2max) and the level of oxidative damage to macromolecules^7,8 as well as muscle mass and the level of ROS in the blood.⁹ On average, the weight of the skeletal muscles¹⁰ and VO_2max¹¹ is higher in men than in women, which may affect gender differences in oxidative stress following maximal-intensity exercise.¹²

However, study results have been divergent.^12–16 The consequence of exceeding exercise intensity corresponding to the second ventilatory threshold (respiratory compensation point – RCP) is the development of decompensated metabolic acidosis,¹⁷ sarcolemma micro-damage, and an increase in intramuscular enzyme activity in the blood, which can lead to inflammation and the intensification of oxidative stress.^8,18–20 To our knowledge, there are no existing studies evaluating the relationship between the level of nitro-oxidative exercise-induced stress in men and women and oxygen uptake and work intensity at RCP, which, along with VO_2max, are indicators of endurance performance.

We put forward the following hypotheses: (1) there is a negative correlation between protein and lipid oxidative damage following maximal-intensity exercise, and oxygen uptake and work intensity (%VO_2max) at the RCP in women and men; (2) nitro-oxidative stress following maximal-intensity exercise results from the intensification of anaerobic processes and muscle fibre micro-damage.

In order to test the hypotheses: (1) nitro-oxidative stress in men and women following exercise at maximal intensity were compared; (2) it was determined whether there is a correlation between post-exercise nitro-oxidative stress, and oxygen uptake, or exercise intensity (%VO_2max) at RCP; (3) it was determined whether there is a correlation between post-exercise nitro-oxidative stress and changes in lactate concentration (Lac), creatine kinase (CK), and lactate dehydrogenase (LDH) in the blood following maximal-intensity exercise.

Nitro-oxidative stress was determined on the basis of post-exercise changes in ox-LDL, 3-NT concentration in the serum, and total antioxidative capacity (TAC) of the plasma.

Material and methods

Study design

The participants performed a graded stress test. Participation in the study was voluntary. The participants were informed about the purpose of the research, familiarized with laboratory procedures, and gave written informed consent to participate in the study. The stress test was performed under the supervision of a sports medicine doctor in the morning, in thermal-neutral conditions. The participants did not perform any intense physical effort 48 hours before or on the day of the test, and neither did they consume any products containing alcohol, caffeine, or other stimulants. The stress test was performed after about eight hours of night sleep.

Participants

The study involved healthy, non-smoking, physically active (light to moderate exercise ≥3 times per week) college aged volunteers (20 women: 21.34 ± 1.57 years and 20 men: 21.97 ± 1.41 years). Medical qualification was carried out (history, physical examination, ECG). No medical contraindications to exercise at maximal intensity were found in any of the volunteers.

Procedures

Somatic measurements

Body height (BH) was measured using the Martin type anthropometer with an accuracy of 0.01 cm (USA). Body mass and body composition fat mass (FM), percentage of body fat (%F) as well as lean body mass (LBM) were determined with the Jawon IOI-353 Body Composition Analyzer (Korea), using bioelectrical impedance analysis (multi-frequency: 5, 50, 200 kHz, tetra-polar technique). Body mass index (BMI) was calculated for each participant. Somatic characteristics of the participants are given in Table 1.

Table 1. Somatic characteristics of study participants (mean ± SE).

Variables	Women (n = 20)	Men (n = 20)	P value
Age (years)	21.34 ± 0.35	21.97 ± 0.32	0.19
BH (cm)	165.99 ± 0.85	181.36 ± 1.16	<0.01
BM (kg)	57.77 ± 1.48	79.00 ± 1.71	<0.01
LBM (kg)	44.54 ± 0.78	65.03 ± 1.38	<0.01
FM (kg)	13.19 ± 0.79	13.96 ± 0.70	0.47
%F (%)	22.48 ± 0.91	17.61 ± 0.72	<0.01
BMI (kg·m−2)	20.94 ± 0.45	23.96 ± 0.35	<0.01

P < 0.01 – significant differences men vs. women.

BH – body height, BM – body mass, LBM – lean body mass, FM – fat mass, %F – percentage of body fat, BMI – body mass index.

Incremental test

The stress test was performed on a treadmill (h/p/Cosmos Saturn COS 10198, Germany) at a 0° angle. After four minutes of warming-up at a speed of 6 km h⁻¹ for women and 7 km h⁻¹ for men, increasing the running speed every 2 minutes by 1.0 km h⁻¹ and 1.2 km h⁻¹ for the men and women, respectively. The exercise test was continued until exhaustion. In the test, VO_2max, maximal heart rate (HR_max) and RCP were determined. Oxygen consumption (VO₂), carbon dioxide production (VCO₂), pulmonary ventilation (V_E), expiratory carbon dioxide concentration (%F_ECO₂), respiratory quotient (RQ), and the ventilatory equivalent ratio for carbon dioxide (V_E/VCO₂) were measured using the Medikro 919 ergospirometer (Finland), starting 2 minutes before the test and until its completion. Averaged values of ergospirometric parameters over 30-second-long periods were registered. Heart rate was recorded during a 5-second interval using the Polar S610i heart rate monitor (Finland).

Criteria for attaining VO_2max

The criteria applied to determine VO_2max were as follows: a plateau in oxygen uptake, a respiratory-exchange-ratio of > 1.1 and attainment of a heart rate within 10 beats per minute of the age-predicted maximum. However, in situations where no plateau was observed, but the rest of the criteria were met, VO₂ peak was taken as the VO_2max.²¹ The maximal oxygen uptake relative to body mass (VO_2max·BM⁻¹) and relative to lean body mass (VO_2max·LBM⁻¹) were calculated.

Criteria for determining RCP

RCP was designated for each participant using the method of respiratory equivalents, similarly as in earlier studies.²² It was assumed that RCP corresponds to the intensity of the exercise, for which the following were determined in total: (a) decrease in %F_ECO₂ after reaching maximal level, (b) rapid nonlinear increase in V_E (second deflection), (c) V_E/VCO₂ ratio reached a minimum and began to increase, (d) rapid nonlinear increase in VCO₂ (second deflection).^17,23

Absolute oxygen consumption at RCP (VO_2RCP) and relative to body mass (VO_2RCP·BM⁻¹), as well as to lean body mass (VO_2RCP·LBM⁻¹) were specified. The RCP was presented as exercise intensity expressed as %VO_2max.

Biochemical analysis

For all markings, blood was taken 5 minutes before and 3 minutes after completion of the graded test after having earlier been in a sitting position, respectively for 10 and 3 minutes.

The Lac was marked in arterialized blood taken from the fingertip and put into micro-tubes containing K2EDTA as an anticoagulant and sodium fluoride as a glycolysis inhibitor, and was immediately centrifuged (MPW 55, Poland). After centrifugation, the concentration of lactate in the plasma was determined using the colorimetric assay method with the L-Lactate Randox enzyme test (UK). Linearity of markings was up to 19.7 mmol L⁻¹. The absorbance was measured at 550 nm using the Evolution 201 Thermo Scientific UV/Vis spectrophotometer (USA). The ox-LDL concentration, 3-NT concentration, and CK as well as LDH activity were marked in the serum of venous blood. TAC was determined in venous plasma. Venous blood was collected from the vein inside the elbow joint and placed into tubes containing coagulation activators or K2EDTA (BD Vacutainer vacuum system (USA)). In order to obtain plasma, the blood was centrifuged immediately after collection, however, in order to obtain serum, the blood was centrifuged after 20 minutes of clotting in room temperature (MPW 351R, Poland). Plasma and serum were stored at −20°C until conduction of the assay and for no longer than 4 weeks. Neither haemolysis nor lipidemia were found in the samples.

The concentration of ox-LDL and 3-NT were determined by enzyme immunoassay tests using the K7810 and K7824 Immundiagnostik test (Germany), respectively. The assays use the ‘sandwich’ technique with two polyclonal antibodies. The concentration of ox-LDL and 3-NT were automatically read from the calibration curves made under the tube analysis conditions. The accuracy of the results was verified on the basis of the readings of trial control concentrations included in the reagent kits.

TAC was marked using the colorimetric assay method with the KC5200 enzyme Immundiagnostik test (Germany). The determination of the TAC was performed by the reaction of antioxidants in the sample with a defined amount of exogenously provided hydrogen peroxide (H₂O₂). Assay sensitivity was 130 µmol L⁻¹. In ox-LDL, 3-NT, and TAC determination, absorbance was measured at 450 nm using the DRG E-lysis MAT 3000 microplate reader (USA).

CK and LDH activity was determined by performing spectrophotometric kinetic measurements using CK BioMaxima (Poland) and LDH BioMaxima (Poland) tests, respectively. Linearity was up to 1000 U L⁻¹ for CK and 2400 U L⁻¹ for LDH. Samples were incubated at 37°C during the reactions and measurements (TPS-1500W Sealed Peltier Recirculator, Thermo Scientific (USA)). The rate of change in absorbance over time, measured at a wavelength of 340 nm (UV/Vis Evolution 201 Thermo Scientific Spectrophotometer (USA)), was proportional to the measured activity of enzymes.

Statistical analysis

The test results are presented as mean values and standard error (mean ± SE). The significance of differences between means was assessed using one- or two-way ANOVA analysis of variance with repeated measurements. In the case of finding significant impact of the main factor (SEX, EXERCISE or SEX and EXERCISE), the significance of differences between specific means using post-hoc analysis (Tukey test) was assessed. Correlations between variables were determined using the Pearson's test. Differences were accepted as statistically significant at P < 0.01. Statistical calculations were performed using Statistica 10.0 (Stat-Soft, Inc., USA).

Results

The absolute and relative VO_2max and VO_2RCP values were significantly higher for men as compared to those obtained for women (P < 0.01). The exercise intensity at the RCP (%VO_2max) did not differ significantly in either of the groups. There were no statistically significant differences in the HR_max and HR at RCP between the two sexes (Table 2).

Table 2. Physical fitness characteristics of study participants (mean ± SE).

Variables	Women (n = 20)	Men (n = 20)	P value
Maximal values of physiological indicators
VO2max (L·min−1)	2.61 ± 0.08	4.51 ± 0.11	<0.01
VO2max·BM−1 (mL·kg−1·min−1)	45.08 ± 0.91	57.38 ± 1.22	<0.01
VO2max LBM−1(mL·kg−1·min−1)	58.38 ± 1.28	69.56 ± 1.37	<0.01
HRmax (b·min−1)	196.25 ± 2.06	199.45 ± 2.12	0.29
Threshold (RCP) values of physiological indicators
VO2RCP (L·min−1)	2.02 ± 0.09	3.45 ± 0.14	<0.01
VO2RCP·BM−1 (mL·kg−1·min−1)	34.84 ± 0.93	43.67 ± 1.42	<0.01
VO2RCP·LBM−1 (mL·kg−1·min−1)	45.17 ± 1.42	52.06 ± 1.80	<0.01
HRRCP (b·min−1)	175.10 ± 2.51	170.25 ± 3.19	0.24
%VO2max	78.46 ± 1.30	75.92 ± 1.52	0.21

P<0.01 – significant differences men vs. women.

VO_2max – maximal oxygen uptake, HR_max – maximal heart rate, RCP – respiratory compensation point, VO_2RCP –oxygen uptake at RCP, HR_RCP – heart rate at RCP, %VO_2max – exercise intensity at RCP.

As a result of the maximal-intensity exercise, a statistically significant and comparable increase in ox-LDL concentration and 3-NT in the blood serum of both groups was found (Table 3).

Table 3. Concentrations of nitro-oxidative stress indicators and lactate, and CK and LDH activity, in the blood of men and women before and after performing the IT (mean ± SE).

	Women (n = 20)		Men (n = 20)		Sex – Exercise interaction
Variables	Before exercise	After exercise	Before exercise	After exercise	P value
ox-LDL (ng·mL−1)	93.5 ± 21.2	101.4 ± 23.3*	116.4 ± 26.2	126.8 ± 28.2*	0.51
3-NT (ng·mL−1)	141.1 ± 15.2	151.9 ± 15.8*	141.9 ± 16.0	155.5 ± 17.0*	0.37
TAC (μmol·L−1)	282.9 ± 10.9	323.0 ± 14.0*	340.7 ± 7.5#	364.3 ± 4.5	0.21
CK (U·L−1)	74.0 ± 43.5	92.2 ± 55.4*	111.8 ± 20.4	130.6 ± 23.1*	0.96
LDH (U·L−1)	237.3 ± 12.0	294.6 ± 16.8*	254.1 ± 16.1	319.3 ± 22.3*	0.63
Lac (mmol·L−1)	1.1 ± 0.1	11.8 ± 0.6*	1.3 ± 0.1	14.7 ± 0.6*(#)	<0.01

Significant differences (P < 0.01) *after exercise vs. before exercise, ^# men vs. women; ox-LDL – oxidized low-density lipoprotein, 3-NT – 3-nitrotyrosine, TAC – total antioxidative capacity, Lac – plasma lactate concentration, CK – creatine kinase activity in serum, LDH – lactate dehydrogenase activity in serum.

Before the exercise, the TAC of plasma in the male group was significantly higher than in women (P < 0.01). After the maximal-intensity exercise, a statistically significant increase in the TAC was observed only in women (Table 3).

After the maximal-intensity exercise, a statistically significant increase in CK and LDH activity in the serum was noticed in both groups. The post-exercise-induced increase in blood plasma Lac was significantly higher in men than in women (Table 3).

Correlations

No statistically significant correlations were found in any of the groups, neither between the absolute values or relative maximal oxygen uptake (VO_2max, VO_2max·BM⁻¹, VO_2max·LBM⁻¹), nor between values regarding oxygen consumption at the level of the RCP (VO_2RCP, VO_2RCP·BM⁻¹, VO_2RCP·LBM⁻¹), exercise intensity at RCP and the changes in ox-LDL, 3-NT and TAC concentrations, induced by maximal-intensity exercise. Only in the group of women was a positive statistically significant correlation found between the post-exercise increase in TAC and changes in CK activity (r = 0.82) and LDH (r = 0.63). No significant correlations were noted in any of the groups between the post-exercise changes in Lac concentration and changes in the concentration of ox-LDL, 3-NT, and TAC.

Discussion

Our research shows that oxidative lipid and protein damage following maximal-intensity exercise is similar in men and women and is not dependent on exercise intensity (maximal and submaximal oxygen uptake). In women, an increase in plasma antioxidative capacity was likely the result of muscle fibre micro-injuries, as evidenced by the positive correlation between TAC and changes in CK and LDH in the blood serum. Due to the higher pre-exercise antioxidative capacity of plasma in men, exercise performed at maximal intensity did not cause significant changes in this indicator among this group.

On the basis of previous research, one unambiguous marker of oxidative stress cannot be indicated.²⁴ Numerous indicators of macromolecule oxidation and antioxidants (enzymatic and non-enzymatic) change their concentrations at various times after exercise. These changes depend on the intensity and time of physical exercise, as well as the age and sex of the subjects. In the research by Michailidis et al.²⁵, in which young men performed a 45-minute submaximal effort (70–75%VO_2max), and then an exercise at an intensity of 90%VO_2max continued until refusal, indicators of oxidative stress were determined before, immediately after, and also 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, and 24 hours after completion of the exercise. The largest increase in the concentration of catalase was found immediately after exercise, and the TAC concentration increased significantly immediately after the exercise, but with the peak value 2 hours after completion of the effort. Immediately after exercise, a significant increase in the concentration of thiobarbituric acid reactive substances (TBARS) and a significant decrease in concentration of reduced glutathione (GSH) were found, with peak changes, respectively, 1 hour and 2 hours after completion of the effort. The greatest changes in oxidized glutathione (GSSG), and the concentration ratios of GSH/GSSG were noted two hours after exercise. Protein carbonyl concentration (PC) increased significantly 30 min after exercise, with the peak concentration 4 hours after the effort.²⁵ Immediately and 30 minutes after exhaustive exercise, there were significant decreases in the concentration of TBARS and a significant increase in PC concentration, similar in young men and women. The activity of superoxide dismutase (SOD) and GSH level significantly increased after exercise in women and did not significantly change in men.²⁶ In both sexes, significant changes in the concentration indicators of oxidative stress occurred 3 minutes after anaerobic exercise.²⁷ Comparing the changes in indicators of oxidative stress after graded exercise in young and older men, oxidative damage to lipids was found only in the older subjects; however, an increase in the activity of antioxidant enzymes was observed only in young men.²⁸ In our research, we identified systemic nitro-oxidative stress within the first minutes after completion of the graded exercise, when substantial disorders of acid-base balance occur, and at the same time, damage to the muscle may be observed, which can affect the severity of oxidative stress.^12,27–29

The obtained results are partly consistent with those of previous studies, which found that changes in the total antioxidant capacity of the blood caused by maximal-intensity exercise are different in men and women.¹² Similar to our study, the pre-exercise TAC level was significantly lower, and the post-exercise change in this indicator was significantly higher, in women.¹² At the same time, in the study conducted by Wiecek et al.,¹² in men a significant shift in prooxidative-antioxidative balance towards oxidation under the influence of exercise without a significant increase in the TAC of plasma was observed. Following maximal-intensity exercise a significant increase in total oxidative status (TOS) as well as the indicator of oxidative stress (TOS/TAC) could only be found in men. Post-exercise changes in the level of TOS in men depend on the absolute values of VO_2max, values of VO_2max relative to LBM and post-exercise changes in Lac.¹² In this study, we did not assess post-exercise changes in TOS, but determined the oxidative damage to proteins and fats based on changes in the concentration of 3-NT and ox-LDL. We found that damage to these macro-molecules of similar severity occurs in women and men following maximal-intensity exercise. Despite significant inter-sex differences in absolute and relative maximal oxygen uptake, we did not find the previously postulated dependence between these values and post-exercise changes in nitro-oxidative stress indicators.^7,8 However, differences between genders in the VO_2max relative to body mass may explain the different TAC levels in men and women found during resting. Previous studies have shown a positive correlation between physical fitness determined by relative values of maximal oxygen uptake and the antioxidative system efficiency in individuals with a proper body composition.^24,30 A significantly lower TAC level in women during resting was also found by Kaikkonen et al.,¹⁵ in contrast to other studies in which no significant differences in antioxidative enzyme activity were found related to the gender of children, adults, or the elderly.³¹ In our study, the concentration of ox-LDL prior to exercise was similar in both groups, which was consistent with the results of other studies³¹ but in contrast with the data of Dopsaj et al.,³² reporting a significantly higher level of oxidized lipid forms in untrained women.

The increase in plasma antioxidative capacity after completion of the graded test may result from the increase in antioxidative enzyme activity.³³ Confirmation of this may be the results of studies in which a significant increase in the activity of SOD was observed only in women upon completion of the graded test.^13,26 Rush et al.¹⁶ noted that, after exercise, glutathione peroxidase (GPx) in blood was significantly higher in women than in men, with no difference in the level of lipid oxidation products (8-iso-PGF2α), which is confirmed by our results. In other studies,²⁰ not only was a decrease found in TAC after graded exercise in men, but also a significant increase in the production of the HO^•, along with a simultaneous increase in total antioxidant capacity.³⁴ In men, evidence of oxidative damage leading to higher concentrations of lipid oxidation products (malondialdehyde) in the plasma, increases in the concentration of oxidized glutathione (GSSG), as well as an increased concentration ratio of oxidized to reduced glutathione (GSSG/GSH) in erythrocytes and protein carbonyl in lymphocytes, accompanied by an increase in antioxidative enzyme activity in the lymphocytes (SOD, GPx, GR) and an increase in the concentration of vitamin E in the lymphocytes, plasma uric acid and the level of SOD in erythrocytes, was found following maximal-intensity exercise.³⁵

The gradually increasing intensity during the incremental test (IT) requires the involvement of anaerobic metabolic ATP re-synthesis pathways, especially after exceeding the RCP.¹⁷ The result of the anaerobic exercise is the formation of ROS and RNS.³⁶ As presented in earlier studies, comparison of the results between men and women following anaerobic exercises points to a significant increase in the concentrations of malondialdehyde with a simultaneous decrease in TAC in the blood of men,³⁷ but also the same changes in TAC, TOS, TOS/TAC, and non-enzymatic antioxidants of low molecular weight in men and women.³⁷ Antonicic-Svetina et al.³⁴ showed that incremental exercise leads to increased HO^• production and its concentration after competition of the exercise is negatively correlated with the running speed at the anaerobic threshold. Our study does not confirm these results. In our research, regardless of gender, we found no correlation between post-exercise changes in nitro-oxidative stress concentration indicators (ox-LDL, 3-NT), changes in TAC and oxygen uptake at the RCP or exercise intensity (%VO_2max) at RCP. In contrast to our study, Antonicic-Svetina et al.³⁴ did not determine the RCP. To our knowledge, we are the first to have studied the correlation between changes in nitro-oxidative stress concentration indicators (ox-LDL, 3-NT) occurring after maximal-intensity exercise in men and women and the exercise intensity at RCP. After exceeding the anaerobic threshold during the graded test, significant increases in the concentration of epinephrine, norepinephrine, and lactate in the blood can be observed.^38,39 After the effort, an increase in the level of IL-1 and TNF-α, indicating activation of inflammatory processes, has been seen.^18,20 In our study, we found a greater increase in the Lac in the group of men. However, this had no effect on the severity of post-exercise prooxidative-antioxidative imbalances, as in previous studies.¹² In our study, after completion of the graded test, a significant increase in both groups was found in CK and LDH in the blood, which are intra-muscle enzymes and biochemical indicators of muscle damage that result in inflammation processes as well as increased production of ROS and RNS.^40–42 In the study by Bouzid et al.²⁸, following graded exercise in a group of young men, a significant increase in antioxidant enzyme activity (SOD, GR, GPx) was observed in the blood, without changing the concentration of MDA. The increase in SOD activity was positively correlated with a significant increase in LDH activity. In contrast to these results, in a group of older men, the graded exercise caused intensification of oxidative stress, and the increase in MDA concentration in the blood was dependent on the increase of CK and LDH activity.²⁸ We conclude that changes in CK and LDH activity were correlated with an increase in the TAC of plasma in women, which may indicate involvement of sarcolemma micro-damage mechanism in the formation of nitro-oxidative stress.

Limitation of the study

Changes in the level of biochemical markers of muscle damage intensify after effort and their levels in the blood can be raised for several days after completion of the exercise, which may be accompanied by a chronic inflammatory state.^40,43 Various indicators of oxidative stress reach their maximum level at different times after the effort.²⁵ The blood samples we collected for biochemical analysis were taken only in the third minute following exercise. In our study, however, we did not specify the post-exercise changes in white blood cells, or changes in levels of pro-inflammatory interleukins. The source of reactive forms of oxygen and nitrogen include adipocytes and macrophages and monocytes in adipose tissue, which are activated by proinflammatory interleukins (TNF-α, IL-1, IL-6).⁴⁴ Given the above limitations, further research should evaluate the post-exercise changes in oxidative stress indicators and indicators of muscle damage over a longer period of time after completion of the exercise. The study should also include overweight, obese, and elderly subjects.

Conclusions

The result of maximal-intensity exercise is a significant increase in the concentration of ox-LDL and 3-NT in the blood serum. Hypothesis 1 was not validated. The gain of ox-LDL and 3-NT is independent of VO_2max, oxygen consumption, and exercise intensity at RCP. This increase of ox-LDL and 3-NT is indicative of similar lipid and protein damage in women and men. Hypothesis 2 was validated in the case of women. A significant increase in the TAC of plasma in women following maximal-intensity exercise is likely the result of muscle fibre micro-injuries.

Disclaimer statements

Contributors First author: study design, data collection, statistical analysis, data interpretation, manuscript preparation. Other authors declare equal contribution: data collection.

Funding This work was supported by University of Physical Education in Krakow, Poland [grant number 267/IFC, grant number 21/BS/IFC/2011]

Conflict of interest None.

Ethics approval The project obtained a positive approval by the Bioethical Commission of the Regional Medical Chamber in Krakow. All test procedures were conducted in accordance with the principles adopted in the Declaration of Helsinki.