Oxidative Stress and Antioxidant Enzymes Activity after Cycling at Different Intensity and Duration

Abstract

This study aimed to compare the effects of intensity (I) and duration (D) on the oxidative stress marker (malondialdehyde, MDA) and the responses of the antioxidant enzymes (catalase, CAT; glutathione peroxidase, GPx; superoxide dismutase, SOD) among sedentary adults. In a crossover design, 25 sedentary adults performed nine cycling exercise sessions with a constant load of 50%, 60%, and 70% VO₂peak for 10-, 20-, and 30-min each. Plasma MDA, CAT, GPx, and SOD activity were measured before and immediately after each exercise session. Results show that MDA concentration and SOD activity increased significantly immediately after exercise at all intensities and durations, except SOD decreased significantly at 70% $\dot{\mathrm{V}}$O₂pk for 30 min. CAT activities also increased significantly after exercise at 50% $\dot{\mathrm{V}}$O₂pk for 10 and 20 min but decreased at 60% $\dot{\mathrm{V}}$O₂pk for 30 min and at 70% $\dot{\mathrm{V}}$O₂pk for all durations. GPx activity decreased significantly after 20 and 30 min at all intensity levels. In conclusion, our results show that cycling at 50%, 60%, and 70% $\dot{\mathrm{V}}$O₂pk for 10, 20, and 30 min increased oxidative stress and antioxidant activities, but with different responses. These findings suggest that the starting exercise intensity for sedentary adults should not exceed 70% $\dot{\mathrm{V}}$O₂pk.

1. Introduction

Regular exercise has beneficial effects on health [1,2] and concurrently increases the production of reactive oxygen species (ROS). Although ROS primarily act as messengers in signal transduction for regulating various cellular functions [3], an imbalance in the equilibrium of ROS and antioxidants is proven to cause higher levels of oxidative stress [4,5]. Previous studies showed that acute exhaustive exercise impaired these health benefits via the rise of ROS and free radical production [6,7].

The increased levels of markers due to oxidative damage to lipids, proteins, and DNA are often said to be caused by the high intensity of exercises [8]. Oxidative stress instigated by exercise is due to the rise in oxygen consumption ($\dot{\mathrm{V}}$O₂) by 10- to 15-fold above the resting level, leading to the increase in oxygen flux by 100-fold higher than the resting value in the contracting skeletal muscles [9]. The oxygen flux’s steep rise is followed by ROS’s overproduction [10]. Subsequently, such a high concentration of ROS overwhelms the antioxidant capacity and affects redox balance [6,8,9].

Oxidative stress associated with exercise could lead to a disruption in cellular homeostasis, such as muscle fatigue [11,12], and after performing a high-intensity exercise, cellular apoptosis [13] and muscle damage ensue [14]. However, exercise also produces the necessary stress to stimulate chronic adaptations that are beneficial [15], including the reduction of inflammatory responses [16,17] and oxidative stress by enhancing the antioxidant system [18]. The degree of oxidation is proportional to the amount of oxidant production contributed by exercise mode, intensity, and duration [19].

Cycling is one of the safest modes of exercise, especially for overweight, obese, and older people for cardiovascular fitness and weight loss training program [20]. It has become popular, especially indoor cycling, with the aid of personal trainers [20], music rhythm [20], and sometimes high energy demand [21]. However, the optimal load (intensity) and duration needed by sedentary adults, especially those unfamiliar with cycling, is still unclear.

As suggested by ACSM [22], the standard guidelines for beneficial exercise effects in healthy adults aged 18–65 years are moderate aerobic physical activity for a minimum of 30 min five days per week or vigorous-intensity aerobic activity for a minimum of 20 min three days per week. According to the intensity classification by Garber et al. [23], low-intensity exercise ranges from 37–45% of $\dot{\mathrm{V}}$O₂max (equivalent to 57–63% HRmax), moderate intensity exercise ranges from 46–63% of $\dot{\mathrm{V}}$O₂max (equivalent to 64–76% HRmax), and high-intensity exercise ranges from 64–90% of $\dot{\mathrm{V}}$O₂max (equivalent to 77–95% HRmax). However, a study by Gaesser and Rich [24] showed that the minimum training-intensity threshold for improving aerobic capacity is at least 45% $\dot{\mathrm{V}}$O₂max.

At these intensities, the production of reactive oxygen species (ROS) has been associated as being a stimulus to physiological endurance adaptations [15,25]. The increased ROS production is maintained by a concomitant increase in antioxidant enzymes such as Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPx). However, the evidence is still limited, particularly regarding different intensity levels and duration of cycling on oxidative stress, especially among sedentary adults. Thus, this study aimed to investigate the optimal intensity and time duration limits of cycling exercise for sedentary healthy adults as the oxidative stress responses seem to be dependent on several factors, including age, gender [25], fitness level and psychological stress [26], nutritional intake [27], training status [28,29], muscle mass involved [24], exercise intensity [27,30], and duration [27]. The age, body composition, fitness level, and nutrient intake were recorded to minimize the variance between recruited subjects. This study employed crossover trials of nine bouts of cycling with intensity ranging from 50% to 70% $\dot{\mathrm{V}}$O₂pk and exercise time of 10, 20, and 30 min that were prescribed explicitly for each healthy and sedentary young male adult.

2. Materials and Methods

2.1. Study Design and Population

A crossover trial was employed in this study to determine the oxidative stress levels and antioxidant enzymes activities after cycling at different intensities and durations. Inclusion criteria were male gender, aged 20–22 years old, and those categorized as sedentary. For the latter, we employed Thivel et al.’s [31] criteria based on performing physical activity between 1–2 times per week for fewer than 20 min each. Those with known co-morbidities such as cardiorespiratory conditions, metabolic diseases, and other pre-existing diagnoses warranting regular prescription of medications, actively smoking, and consuming alcohol regularly were excluded from the study. Eligible subjects were interviewed and measured for demographic information, basic body parameters, total nutrient intake, and four primary outcomes, as explained in the following section. Nutrient intake was analyzed using a modified Food Frequency Questionnaire by Komposisi Nutrisi Makanan Malaysia and Diet 4 software.

A total of 60 male subjects aged 20 to 22 years old were screened for eligibility, and 31 of them fulfilled the criteria. As presented in

Table 1. Characteristics of all subjects and their nutritional intake pre- and post-training.

Characteristics	Mean ± SE
Age	20.8 ± 0.45 years
Bodyweight	58.8 ± 2.39 kg
Height	167.5 ± 1.12 cm
BMI	20.9 ± 0.84 (kg/m−2)
Fat Mass	10.8 ± 1.20 kg
Body Fat	17.4 ± 1.25%
Measured $\dot{\mathrm{V}}$O2pk	36.6 ± 1.17 (mL/min/kg)
Predicted $\dot{\mathrm{V}}$O2pk	48.1 ± 1.10 * (mL/min/kg)
Nutrient level	Pre-training	Post-training
Calorie	1853 ± 70 kcal	1771 ± 85 kcal
Protein	112 ± 3 g	118 ± 2 g
Carbohydrate	281 ± 18	272 ± 16
Fat	38 ± 3	39 ± 2
Retinol	197 ± 30 µg	193 ± 32 µg
Carotene	1511 ± 423 µg	1482 ± 512 µg
Ascorbic Acid	83 ± 12 mg	88 ± 13 mg

N = 25; BMI—Body Mass Index; $\dot{\mathrm{V}}$O₂pk —Peak Oxygen Consumption; * measured $\dot{\mathrm{V}}$O₂pk significantly different from predicted $\dot{\mathrm{V}}$O₂pk at p < 0.05.

, the measured $\dot{\mathrm{V}}$O₂pk (36.6 ± 1.17 mL/min/kg) was significantly lower than the predicted $\dot{\mathrm{V}}$O₂pk (48.1 ± 1.10) at p < 0.05, indicating subjects are sedentary [32]. Only 25 subjects completed the exercise program, which was based on G*Power. This figure fulfilled the calculated sample size based on the power of 1− β = 0.95. Subject dropout was caused by a loss of interest to continue and the inability to perform the exercise with the workload given.

Table 1 summarizes the characteristics of all subjects. The body mass index (BMI) ranged between 18.5 to 24.9 kg/m⁻². The mean percentage of subjects’ body fat (17.4 ± 1.25%) was consistent in the fair category [33]. The total intake of calories, protein, carbohydrate, fat, retinol, carotene, and ascorbic acid before and three months after the nine exercise bouts did not differ significantly.

2.2. Outcome Variables

Each subject was assessed for four primary outcomes: (1) peak aerobic capacity, (2) maximum heart rate, (3) oxidative stress status, and (4) erythrocyte antioxidant enzymes analysis. Peak aerobic capacity and maximum heart rate was assessed at pre-exercise and post-exercise with a graded exercise cycling test. A peripheral blood sample was obtained from the forearm vein of each subject into a BD Vacutainer^® (BD Life Sciences, New Jersey, USA) tube coated with K₂EDTA before and immediately after each exercise bout. Blood samples were centrifuged at 3000 rpm for 10 min at 4 °C to separate plasma from red blood cell pellets. The resulting plasma and red blood cell samples were frozen and stored at −80 °C.

Plasma malondialdehyde (MDA) was used as an oxidative stress status marker, and the levels were determined according to Pilz et al. [34]. The intracellular antioxidant markers were measured using erythrocyte antioxidant enzymes activities, including superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). Activity for one unit of SOD was designated as the amount of haemoglobin that inhibits the rate of nitro blue tetrazolium reduction by 50% [35]. GPx activity was determined spectrophotometrically by the method of Paglia and Valentine [36]. H₂O₂ was added to a medium containing phosphate buffer, EDTA, NaN₃, NADPH, GSH and erythrocyte hemolysate, followed by measuring the change in absorbance of the system at 340 nm. These antioxidant enzymatic activities were expressed relative to the haemoglobin concentration. CAT activity was quantified using a spectrophotometer based on the method previously described by Aebi [37]. The decomposition of H₂O₂ by the CAT enzyme was followed directly by the decrease in absorbance at 240 nm. The difference in absorbance per unit time was measured as CAT activity. All samples were performed in duplicate.

2.3. Exercise Bouts

After the adequate screening, eligible subjects completed a graded exercise cycling test to measure peak aerobic capacity ($\dot{\mathrm{V}}$O₂pk) and maximum heart rate (HRmax) for determining the subsequent workloads for nine exercise bouts. $\dot{\mathrm{V}}$O₂pk was measured through a one-minute incremental exercise protocol on a cycle ergometer (Ergometrics 900, Ergoline: Bitz, Germany) as previously described [38]. While exercising, Cortex Metamax 3B (Leipzig, Germany) measured the gas exchange while heart rate was recorded with Polar Electro, Inc. (Woodbury, NY, USA), heart monitor. Each subject cycled with unloaded pedaling for 3 min, followed by exercising at the incremental workload. The subject was encouraged to maintain cycling exercise until they could not sustain the workload for more than 30 s, or the cycling frequency between 50–60 rpm could not be maintained, or when the subject decided to terminate the exercise. Additional criteria of having a respiratory exchange ratio of more than 1.1 were used to ensure that maximal O₂ uptake was reached. Results determined each subject’s corresponding workload for 70%, 60%, and 50% of $\dot{\mathrm{V}}$O₂ peak ($\dot{\mathrm{V}}$O₂pk).

The study’s main objective was to determine the oxidative stress levels after cycling at different intensities and durations through a crossover study design. Subjects underwent nine exercise bouts with different intensities and durations, one after another, in random order; either the 50% $\dot{\mathrm{V}}$O₂pk for 10 min; 60% $\dot{\mathrm{V}}$O₂pk for 10-min; 70% $\dot{\mathrm{V}}$O₂pk for 10 min; 50% $\dot{\mathrm{V}}$O₂pk for 20-min; 60% $\dot{\mathrm{V}}$O₂pk for 20-min; 70% $\dot{\mathrm{V}}$O₂pk for 20-min; 50% $\dot{\mathrm{V}}$O₂pk for 30-min; 60% $\dot{\mathrm{V}}$O₂pk for 30-min; or 70% $\dot{\mathrm{V}}$O₂pk for 30-min. Subjects performed exercise at a constant predetermined work rate for each intensity throughout the duration without an increase in $\dot{\mathrm{V}}$O₂. The exercises in this study were performed in a temperature-controlled laboratory between 18 °C to 20 °C to ensure that the measured $\dot{\mathrm{V}}$O₂ and HR for each exercise bout could be maintained.

A constant load exercise was used in this study. As a general concept, consistent load exercise is performed at a constant workload, without or with minimum heart rate and $\dot{\mathrm{V}}$O₂ fluctuations throughout the exercise [39]. The exercise workload for corresponding exercise intensity (% $\dot{\mathrm{V}}$O₂pk) was based on target $\dot{\mathrm{V}}$O₂ and target HR (HR_target) using results of the $\dot{\mathrm{V}}$O₂pk and maximal HR (HRmax) in graded exercise testing [40].

The $\dot{\mathrm{V}}$O₂ and HR were monitored to ensure that subjects exercised at the given intensity. To minimize the residue effect of exercise bouts and ensure that the findings observed are due to the effect of different exercise intensities and duration and not others, subjects were given three days of rest (wash-out period) after the maximal exercise test and three days intervals in between exercise bouts. The usual physical activity and nutritional intake levels were maintained throughout the experimental period. This study was conducted according to the guidelines of the Declaration of Helsinki and following the specifications approved by the Ethical Committee of University Kebangsaan Malaysia, Kuala Lumpur (Ref. no.: UKM 1.5.3.5/244/PPP2). All subjects had agreed to participate in this study by providing written informed consent.

2.4. Statistical Analysis

All data are expressed in mean ± standard error (SE). Data were analyzed for normality distribution using the Shapiro–Wilk test. Analysis of variance (ANOVA) with repeated measures was performed for all exercise bouts by all subjects, with time (pre-exercise and post-exercise), exercise intensity, and duration as the within-subject factors. The data’s homogeneity of covariance or sphericity assumption was confirmed using the Mauchly test. Homogeneity of variances was tested for every level’s sample size. The sphericity assumptions for SOD, CAT, and GPx data sets were maintained at p < 0.05. All statistical analysis was performed using Statistical Packages for Social Sciences (IBM SPSS Statistics for Windows, Version 22.0, IBM SPSS Inc, Armonk, NY, USA).

3. Results

3.1. VO₂ Level and HR

There were no significant differences in measured $\dot{\mathrm{V}}$O₂ and HR between the 10-, 20-, and 30-min for each exercise intensity.

Table 2. Oxygen usage and heart rate (HR) responses at different intensity percentages and duration of exercise.

Duration (Mins)	Intensity (%)	$\mathbf{Measured} \dot{\mathrm{V}}$O2 (Mean ± SE)	Exercise HR (Mean ± SE)
10	50% $\dot{\mathrm{V}}$O2pk	20.30 ± 1.088	127.56 ± 3.586
20		20.31 ± 0.983	126.36 ± 3.298
30		20.37 ± 1.030	126.28 ± 3.246
10	60% $\dot{\mathrm{V}}$O2pk	24.15 ± 1.001	148.68 ± 3.718
20		24.05 ± 0.926	148.08 ± 3.577
30		24.35 ± 1.004	149.56 ± 3.663
10	70% $\dot{\mathrm{V}}$O2pk	27.58 ± 1.125	166.64 ± 4.206
20		27.88 ± 1.147	167.64 ± 4.233
30		27.90 ± 1.056	169.32 ± 3.945

% $\dot{\mathrm{V}}$O₂pk of peak oxygen consumption, N number of subjects = 25, % changes percentage changes.

summarizes the mean measured $\dot{\mathrm{V}}$O₂ and HR for each exercise duration and intensity.

3.2. Oxidative Stress and Antioxidant Enzymes Responses

We observed that MDA concentration increased significantly immediately after exercise at all intensity levels and durations (

Table 3. Changes in antioxidant enzymes activities and MDA concentration after exercise intervention at different intensities and duration.

I % $\dot{\mathbf{V}}$O2pk	D mins		SOD (Unit/mgHb)	CAT (u/s/mgHb)	GPx (Unit/min/mgHb)	AE (Unit/min/mgHb)	MDA (nmol/mL)
		(Mean ± SEM)
50%	10	Pre	0.83 ± 0.049	0.29 ± 0.019	6.009 ± 0.0002	3.172 ± 0.3333	9.23 ± 0.357
		Post	1.41 ± 0.082 *	0.36 ± 0.018 *	5.621 ± 0.0001	4.009 ± 0.2914	10.28 ± 0.300 *
		% changes	93.3 ± 22.02	44.6 ± 14.62	−4.5 ± 3.00	61.52 ± 21.804	12.7 ± 1.81
60%	10	Pre	0.91 ± 0.096	0.40 ± 0.022	5.507 ± 0.0001	2.447 ± 0.2873	8.41 ± 0.381
		Post	1.28 ± 0.044 *	0.41 ± 0.021	5.112 ± 0.0001	3.248 ± 0.1839 *	9.97 ± 0.322 *
		% changes	139.3 ± 47.45	4.9 ± 4.77	−4.3 ± 4.95	147.51 ± 51.332	21.5 ± 3.79
70%	10	Pre	1.14 ± 0.048	0.34 ± 0.012	7.281 ± 0.0004	3.413 ± 0.1889	10.01 ± 0.360
		Post	1.68 ± 0.069 *	0.31 ± 0.018 *	6.491 ± 0.0004	5.679 ± 0.4152 *	12.98 ± 0.356 *
		% changes	52.6 ± 8.72	−8.6 ± 3.49	−2.9 ± 8.27	72.07 ± 11.272	32.1 ± 3.91
50%	20	Pre	1.18 ± 0.052	0.45 ± 0.027	6.941 ± 0.0002	2.842 ± 0.2218	9.10 ± 0.265
		Post	1.54 ± 0.092 *	0.49 ± 0.029 *	6.058 ± 0.0001 *	3.324 ± 0.3163	10.31 ± 0.289 *
		% changes	34.7 ± 8.74	12.8 ± 3.79	−10.4 ± 3.75	21.59 ± 7.984	14.7 ± 3.54
60%	20	Pre	1.19 ±0.082	0.41 ± 0.019	6.755 ± 0.0001	2.994 ± 0.2171	8.58 ± 0.383
		Post	1.54 ± 0.051 *	0.42 ± 0.027	6.303 ± 0.0001 *	4.189 ± 0.4255 *	10.57 ± 0.255 *
		% changes	44.8 ± 12.16	3.8 ± 6.17	−5.7 ± 2.69	49.34 ± 12.552	30.4 ± 7.45
70%	20	Pre	1.15 ±0.060	0.54 ± 0.031	6.573 ± 0.0002	2.308 ± 0.1951	9.37 ± 0.341
		Post	1.51 ± 0.073 *	0.49 ± 0.037 *	6.079 ± 0.0003 *	3.496 ± 0.3218 *	13.20 ± 0.249 *
		% changes	40.5 ± 10.79	−9.7 ± 3.77	−7.1 ± 3.32	59.59 ± 13.852	44.6 ± 4.88
50%	30	Pre	1.01 ± 0.067	0.40 ± 0.023	7.363 ± 0.0001	2.617 ± 0.1938	8.49 ± 0.277
		Post	1.21 ± 0.066 *	0.41 ± 0.026	6.995 ± 0.0001 *	3.070 ± 0.2187	10.16 ± 0.261 *
		% changes	30.8 ± 10.38	4.9 ± 5.38	−4.1 ± 2.26	28.35 ± 10.470	21.3 ± 3.26
60%	30	Pre	1.73 ± 0.112	0.37 ± 0.024	5.948 ± 0.0001	5.319 ± 0.5969	9.06 ± 0.324
		Post	1.99 ± 0.134 *	0.34 ± 0.021 *	5.336 ± 0.0001 *	6.373 ± 0.5307 *	11.88 ± 0.365 *
		% changes	20.2 ± 8.07	−6.5 ± 2.93	−10.3 ± 1.88	32.55 ± 10.239	32.3 ± 2.99
70%	30	Pre	1.17 ± 0.053	0.33 ± 0.019	8.031 ± 0.0002	3.835 ± 0.3066	8.36 ± 0.369
		Post	0.87 ± 0.039 *	0.29 ± 0.017 *	7.610 ± 0.0001 *	3.076 ± 0.1973 *	13.85 ± 0.302 *
		% changes	−22.7 ± 4.10	−7.8 ± 3.81	−4.8 ± 1.31	−11.53 ± 7.246	72.5 ± 7.41
ANOVA	IXD		F = 1.68 df = 2.055	F = 3.38 * df = 2.146	F = 0.524 df = 1.99	F = 2.117 df = 1.982	F = 4.28 * df = 2.989
	I		F = 5.58 * df = 1.289	F = 14.24* df = 1.619	F = 0.152 df = 1.48	F = 3.035 df = 1.32	F = 54.24 * df = 2
	D		F = 11.82 * df = 1.166	F = 5.96 * df = 1.347	F = 0.65 df = 1.49	F = 11.63 * df = 1.201	F = 8.62 * df = 2

% $\dot{\mathrm{V}}$O₂pk: % of peak oxygen consumption, N number of subjects = 25, % changes percentage changes, * significantly different from pre-exercise at p < 0.05; I = intensity; D = duration.

). SOD activity also rose immediately after completing all exercise bouts, except it decreased significantly at 70% $\dot{\mathrm{V}}$O₂pk for 30 min. CAT activities also increased substantially after exercise at 50% $\dot{\mathrm{V}}$O₂pk for 10 and 20 min but decreased after exercise at 60% $\dot{\mathrm{V}}$O₂pk for 30 min duration (Table 3). As exercise intensity intensified to 70% $\dot{\mathrm{V}}$O₂pk, the CAT activities significantly reduced after exercise at all durations.

Similarly, GPx activity decreased significantly after 20 and 30 min at all intensity levels (Table 3). The ratio of activities of antioxidant enzymes was calculated using $AE=\frac{SOD}{GPx+CAT}$ [41]. In contrast, the antioxidant enzyme activity ratio only increased after exercise at 60% and 70% $\dot{\mathrm{V}}$O₂pk for all durations, except it decreased at 70% $\dot{\mathrm{V}}$O₂pk for 30 min (Table 3).

The percentage increment of MDA continued to increase (Figure 1), and even the percentage increment of MDA increased exponentially after exercise at 70% $\dot{\mathrm{V}}$O₂pk for 30 min. However, the percentage increment of CAT decreased in response to the increment of both intensity and duration of exercise (Figure 2). The percentage changes of CAT activities were positive across all exercise intensity and duration except at 70% $\dot{\mathrm{V}}$O₂pk for all durations and at 60% $\dot{\mathrm{V}}$O₂pk for 30 min. As for GPx, the percentage changes in GPx are low and did not vary after exercise at different intensities and duration (Figure 3).

As opposed to CAT, the percentage changes of AE and SOD exhibited a similar pattern, with positive percentage changes after all exercise bouts except negative percentage change after exercising at 70% $\dot{\mathrm{V}}$O₂pk for 30 min (Figure 4 and Figure 5). Furthermore, the highest percent increase of SOD and AE is after exercise for 10 min at all percentage intensities of 50%, 60%, and 70% $\dot{\mathrm{V}}$O₂pk. Figure 4 and Figure 5 showed the percentage changes of SOD and AE decreased with exercise duration but increased with exercise intensity.

Repeated measures ANOVA result analysis (Table 3) summarizes the interaction effect of exercise intensity and duration on percentage changes of SOD, CAT, GPx, AE, and MDA levels. A higher F value suggested that exercise intensity is the major controlling factor for MDA (p < 0.01) and CAT (p < 0.01). This implies exercise intensity is a more substantial stimulus than exercise duration to induce MDA concentration and CAT responses. Conversely, the F value of exercise duration is higher than exercise intensity for SOD (p < 0.01) and AE (p < 0.01). This indicates that exercise duration is the primary determinant factor of SOD and AE.

4. Discussion

Physical exercise such as walking or resistance exercise improves cardiovascular and muscular endurance [42] and anxiety [43] and reduces the risk of metabolic diseases [44]. These adaptations are optimally seen following moderate-intensity training [45]. In addition, training at any intensity above 60% of $\dot{\mathrm{V}}$O₂max, including high intensity, is likely to improve maximal oxygen uptake in healthy adults [46]. Hence, high-intensity exercise also can reduce the risk of developing cardiovascular diseases and all-cause mortality by increasing the $\dot{\mathrm{V}}$O₂max [47]. However, exercise at high-intensity loads may adversely affect tissues and contribute to overuse injuries [48]. Quindry J.C. et al. [48] also found that high-intensity endurance exercise increases oxidative stress levels in the blood in untrained subjects. The amount of oxidant production was not only contributed by intensity and duration of exercise but also by exercise mode [49].

Cycling, as one of the non-weight-bearing exercise modes, is becoming increasingly popular, especially in weight loss programs. Exercise results in a 1- to 3-fold increase in superoxide during muscle contraction [50]. Previous studies have reported increased oxidative stress for healthy and diseased subjects following single bouts of exercise [51,52]. A study by McAllister et al. [27] revealed that the increase in oxidative stress among trained subjects occurred following short duration, 30 and 60 min of moderate intensity exercise. This is consistent with the present study, which showed increased oxidative stress, as measured by MDA, immediately following each exercise bout of moderate-to-high intensity and short-to-moderate duration. According to Radak et al., the magnitude of oxidative damage due to exercise depends on oxygen consumption, ROS production rate, and the balance between antioxidants and ROS [10].

In contrast, Lu et al. [8] found that the MDA was not increased after cycling for 30 min at an intensity of 70% $\dot{\mathrm{V}}$O₂max among 10 trained male athletes. This might be stipulated by active participants or trained athletes possessing greater protection against ROS oxidation than sedentary participants [10]. A high level of protection against ROS reduces lipid peroxidation (MDA). Perrone et al. [53] also suggested that the oxidative stress produced from exercise depends on the capacity and the adaptability of the antioxidant defense in the body.

This study also discovered that even low intensity, as in 50% $\dot{\mathrm{V}}$O₂pk, increased the MDA level in sedentary healthy young adults. Thus, we can conclude that the exercise threshold for stimulating oxidative stress during cycling is between 10 to 30 min of 50% to 70% $\dot{\mathrm{V}}$O₂pk in sedentary young adults. However, 30 min duration at 70% $\dot{\mathrm{V}}$O₂pk has caused a marked increase in MDA. The intensity of exercise exaggerates the respiration rate [54], leading to the amplification of oxygen consumption and electron transport chain reaction [10]. It is estimated that the whole body’s oxygen consumption may increase by 10–20 fold [55], while a staggering figure of up to 100–200 fold is seen in the exercising muscles [10].

An increase in MDA following exercise is accompanied by increased antioxidant responses [56,57] that were also discovered in the present study, particularly AE, SOD, and CAT. Antioxidant enzyme activities increased following exercise to counteract the rise in ROS production [6,19,52,58]. The present study discovered that SOD activities increased immediately after cycling at all intensities and duration, resulting in the overproduction of H₂O₂ (hydrogen peroxide) and subsequently overwhelming the GPx and CAT enzymes.

This finding implies that an increase in exercise duration would more highly activate oxidative muscle fibers—type I and type IIa muscle fibers leading to a more significant increase in the SOD activity [59]. Thus, excessive H₂O₂ produced from the dismutation reaction of superoxide inhibited SOD and CAT enzymes by changing the redox condition in the cell and changes in the antioxidant enzyme’s catalytic center [60,61].

SOD is also sensitive to the overproduction of superoxide and hydrogen peroxidase [61], a fact reflected in this study, whereby the SOD significantly decreased after exercising at 70% $\dot{\mathrm{V}}$O₂pk for 30 min. According to Garaiová et al. [41], AE changes the equilibrium between the formation of hydrogen peroxide from superoxide dismutation and its decomposition by other enzymes (GPx, CAT) in erythrocytes. The reduction of AE and the increase of MDA in this study showed that exercising at 70% $\dot{\mathrm{V}}$O₂pk for longer than 20 min induces oxidative stress. These findings suggest that these results may support the theory that the contribution of antioxidant enzyme disequilibrium from oxidative stress during exercise is secondary to limited CAT activity and most likely due to an insufficient increase in the GPx activity. In other terms, oxidative stress is initiated by an imbalance in the activities of antioxidant enzymes, SOD, against GPx and CAT. It can be postulated that exercising at a higher intensity and for a longer duration is associated with the overproduction of free radicals (Figure 6).

Another possible factor that affects CAT activity is blood glucose. It is stipulated that the increase in blood glucose inhibits CAT [62]. The exponential rise of adrenalin and noradrenalin hormones with intensity was much faster than the increment seen with exercise duration. The latter only showed a linear relationship [63]. Both adrenalin and noradrenalin hormones stimulate β-adrenergic receptors in the pancreas and increase glucagon secretion [64]. Resultant high glucagon in plasma increases the blood glucose level through the mobilization of free fatty acid (FFA) from adipose tissue, mobilization of glucose from the liver, and an increase of gluconeogenesis [65].

It appears that the sensitivity of cells to free radicals depends on the equilibrium between the formation of hydrogen peroxide from superoxide in the dismutation reaction catalyzed by SOD and its degradation by GPx and CAT rather than on the activities of individual antioxidant enzymes [66]. In this study, the AE was observed to increase significantly only after exercise at 60% and 70% $\dot{\mathrm{V}}$O₂pk for 10-, 20-, and 30-min, except for 70% $\dot{\mathrm{V}}$O₂pk intensity in 30-min duration, where the ratio was reduced. These results are primarily in agreement with a previous study by Georgakouli et al. [67] that observed a significant elevation of plasma total antioxidant capacity among healthy individuals after 30 min at 50–60% of the heart rate reserve on a cycle ergometer. The increment in plasma MDA with intensity and duration found in this study suggests that the balance of oxygen metabolism is compromised during exercise. Similarly, this finding supports the hypothesis of exercise-induced oxidative stress among sedentary adults [8,44,59,68].

The reduction in all antioxidant enzyme activities (SOD, CAT and GPx) leading to high oxidative damage showed an utmost increment in MDA. Inadequate protection by antioxidants causes cellular damage to increase due to increased lipid peroxidation and oxidative stress [53,69]. In the present study, lipid peroxidation increased with the intensity and duration of cycling exercise. When the intensity and duration of exercise are increased, oxygen consumption [70], metabolic stress [71], metabolism rate [72], and mechanical stress [72,73] will escalate, leading to a higher production of ROS. Sedentary adults with low economical cycling efficiency will not only encounter higher mechanical stress but also deal with high energy demand, especially during cycling at a high intensity.

R. de Melo dos Santos et al. [74] found that cycling requires the subject to continuously adjust the force produced and its timing relative to the pedal position to obtain specific self-selected pacing. Sedentary adults generally use small muscles such as the gastrocnemius and the foot as prime movers. Poor pedaling technique with low ankle ROM produces higher torques to pedal at the same power output and higher metabolic cost of pedaling in sedentary subjects [75]. This finding supports Scribbans et al.’s [46] suggestion that the increment in $\dot{\mathrm{V}}$O₂max using higher intensity training with shorter training bouts and lower training volumes is comparable with moderate intensity training.

Based on the bell-shaped hormesis curve, the optimum level of ROS produced by exercise promotes significant physiological benefits. However, after reaching the highest physiological benefit, a further increase in ROS production can cause tissue damage and reduce functional adaptation [11]. Given that sedentary adults possess a lower cardiopulmonary capacity and poor cycling efficiency, thirty minutes of high-intensity cycling exercise reaches the level of ROS production and higher oxidative level, causing the downward slope among sedentary healthy adults. This study supports that high intensity exercise results in extreme oxidant-mediated damage in cells and a decrease in the antioxidant capacity of the muscles [76].

This study also revealed that the intensity of 70% $\dot{\mathrm{V}}$O₂pk generated the highest stressor stimulus for pro-oxidative than the lower-intensity exercises for all 10-, 20-, and 30-min exercise durations. Exercise at 70% $\dot{\mathrm{V}}$O₂pk has affected the redox balance by producing more ROS which increased the SOD activity and accumulation of H₂O₂. The H₂O₂ build-up following exercise at 70% $\dot{\mathrm{V}}$O₂pk for 30 min has also inhibited SOD and decreased its activity. Thus, this study discloses that moderate intensity is more effective in controlling the response of antioxidant enzymes. This study also suggests that the maximum duration of cycling at a high intensity should not exceed 20 min to produce an optimal amount of oxidative stress needed for adaptation. Johnson et al. [77] have found that the duration required for exercise with a threshold intensity of 50% to 70% $\dot{\mathrm{V}}$O₂pk in trained subjects is between 20 and 60 min.

This study also suggests that high-intensity exercise achieves its optimal potency if the exercise performed is not more than 20 min. Considering high-intensity exercise for a short duration is time-efficient compared to moderate and low intensity [78], we recommended that cycling at a high intensity (at and above 70%$\dot{\mathrm{V}}$O₂pk) should be performed for a short duration or an interval training. While our data support claims regarding the efficiency and potency of high-intensity training, future research is needed to examine the impact of high exercise intensity on improvements in $\dot{\mathrm{V}}$O₂max and how the high-intensity exercise achieves its effectiveness.

Moreover, our findings indicate that free radicals produced during exercise at 70% $\dot{\mathrm{V}}$O₂pk for 30 min have exceeded the capacity of the antioxidant enzyme. This finding supports the suggestion [79] for accumulating at least 30 min of exercise daily at moderate intensity to maintain cardiovascular fitness and reduce potential risks of non-communicable diseases. This finding contradicts previous studies demonstrating increased oxidative stress after moderate-intensity exercise among young, healthy male subjects [30]. Moderate-intensity exercise is thought to confer beneficial effects, but prolonged exercise leads to elevated ROS production at higher exercise intensities [77,80]. However, a significant plasma total antioxidant capacity elevation was observed in a healthy untrained male adult after cycling for 30 min at 70% of the maximum workload [81].

For any exercise to deliver the expected health benefit, there should be an optimal level of ROS produced during exercise that may induce favorable adaptations following repeated exposure [15], including increased expression of antioxidant enzymes over time, such as superoxide dismutase and catalase [82]. However, increasing oxidative stress above the optimal level may compromise health and performance. Too much ROS might impair antioxidant defense capacities leading to substantial cell damage [83,84]. Prolonged and irreparable oxidative damage could predispose to diseases such as neurodegeneration and cardiovascular [80].

Overall, exercise intensity should not continuously exceed 70% $\dot{\mathrm{V}}$O₂pk for more than 20 min. However, this was limited to acute changes in oxidative stress levels and antioxidant enzymes activity among healthy, sedentary young adult males. Which, the chronic state of oxidative stress produced by exercise at intensity not more than 70% $\dot{\mathrm{V}}$O₂pk, mediating the adaptation of antioxidant enzymes was not investigated in this study. This study also does not examine the effect of post-exercise rest at different intensities and durations, which also contributes to oxidative stress levels. Another limitation of this study is that it does not address the cycle efficiency factor, which also contributes to oxygen consumption during exercise.

5. Conclusions

In conclusion, cycling at 50%, 60%, and 70% $\dot{\mathrm{V}}$O₂pk for 10, 20, and 30 min can potentially increase oxidative stress and antioxidant activities acutely, but with different responses. These findings confirm that oxidative stress markers responses seem to be dependent on the duration of moderate intensity of exercise and may help in the prescription of more effective and less harmful effects of exercise-induced oxidative stress among this population. For sedentary adults, a short duration of high-intensity exercise can be proposed for initial exercise levels. Hence, we suggest that the starting exercise intensity for sedentary adults should not be more than 70% $\dot{\mathrm{V}}$O₂pk.

These findings further emphasize the need to achieve optimal exercise intensity and duration and provide physical trainers, exercise enthusiasts, and clinical practitioners with practical settings. Here, an optimal exercise intensity and duration bring beneficial health outcomes. Concurrently, works should evaluate the long-term effect of this study’s optimal intensity and duration. An investigation should be conducted to determine the chronic state of oxidative stress and antioxidant enzyme responses concerning exercise intensity and duration. The existing literature is yet to demonstrate the physiological mechanism that underpins the oxidative process during exercise, especially when the intensity or duration is increased. Here remain the opportunities for future research to evaluate the differences in the production pathways of ROS and free radicals based on different exercise intensities and durations.