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bianca.campagnaro@uvv.br

Vila Velha University (UVV)

(Brasil)

Reception: 10/21/2024 - Acceptance: 09/24/2025

1st Review: 07/24/2025 - 2nd Review: 09/20/2025

Accessible document. Law N° 26.653. WCAG 2.0

Suggested reference: Gouveia, ACGC, Affonso, HO, Coco, LZ, Bis, ARF, Santos, MC, Costa, MAS, Morra, EA, Zaniqueli, DA, Aires, R., Pereira, TMC, & Campagnaro, BP (2025). Lymphocyte Apoptosis and Oxidative Stress Following Short-Term Beach Volleyball Competition in Elite Athletes. Lecturas: Educación Física y Deportes, 30(330), 113-128. https://doi.org/10.46642/efd.v30i330.7953

 

Abstract

    Continuous, strenuous, and prolonged exercise has a well-documented impact on the immune system. This study aimed to verify whether beach volleyball can cause lymphocyte apoptosis and a concomitant increase in oxidative stress. Apoptotic leukocyte cells with FITC-labeled anti-annexin V antibodies and the detection of nuclear propidium iodide, the availability of hydrogen peroxide (H₂O₂) through the oxidation of 2′,7′-dichlorofluorescin diacetate, and the total concentration of antioxidants by the iron reduction antioxidant power assay, before and after a short beach volleyball tournament with 20 elite athletes, were evaluated. The median and interquartile range of H₂O₂ in arbitrary units increased from 2886 before competition to 10402 after competition for female group, and from 2711 to 11154, for male group. The percentage of apoptosis-positive cells increased from 0.7 to 3.9 for female group and from 0.7 to 4.0 for male group. The total concentration of antioxidants did not change, while HDL cholesterol increased in both groups at the end of the competition. Concomitant apoptosis and increased H₂O₂ production in lymphocytes suggest oxidative stress-mediated apoptosis. Antioxidant defense is not activated immediately to restore the redox balance of immune cells, while improved lipid profile suggests antioxidant protection for blood vessels.

    Keywords: Physical exercise. Lymphocyte apoptosis. Oxidative stress. Antioxidant defense. Biomarkes.

 

Resumo

    O exercício contínuo, extenuante e prolongado tem um impacto bem documentado no sistema imunológico. Este estudo teve como objetivo verificar se o voleibol de praia pode causar apoptose de linfócitos e concomitante aumento do estresse oxidativo. Células leucocitárias apoptóticas com anticorpos anti-anexina V marcados com FITC e detecção de iodeto de propídio nuclear, disponibilidade de peróxido de hidrogênio (H₂O₂) por meio da oxidação do diacetato de 2',7'-diclorofluorescina e concentração total de antioxidantes pelo ferro foram avaliados ensaios de redução do poder antioxidante, antes e após um torneio curto de vôlei de praia com 20 atletas de elite. A mediana e o intervalo interquartil de H₂O₂ em unidades arbitrárias aumentaram de 2.886 antes da competição para 10.402 após a competição para p grupo feminino, e de 2.711 para 11.154, no grupo masculino. A percentagem de células positivas para apoptose aumentou de 0,7 para 3,9 no grupo das mulheres e de 0,7 para 4,0 no grupo dos homens. A concentração total de antioxidantes não se alterou, enquanto o colesterol HDL aumentou em ambos os grupos ao final da competição. A apoptose concomitante e o aumento da produção de H₂O₂ nos linfócitos sugerem apoptose mediada por estresse oxidativo. A defesa antioxidante não é ativada imediatamente para restaurar o equilíbrio redox das células imunológicas, enquanto a melhora do perfil lipídico sugere proteção antioxidante para os vasos sanguíneos.

    Unitermos: Exercício físico. Apoptose de linfócitos. Estresse oxidativo. Defesa antioxidante. Biomarcas.

 

Resumen

    El ejercicio continuo, extenuante y prolongado tiene un impacto bien documentado en el sistema inmunitario. Este estudio tuvo como objetivo verificar si el vóleibol de playa puede causar apoptosis linfocitaria y un aumento concomitante del estrés oxidativo. Se evaluaron las células leucocitarias apoptóticas con anticuerpos anti-anexina V marcados con FITC y la detección de yoduro de propidio nuclear, la disponibilidad de peróxido de hidrógeno (H₂O₂) mediante la oxidación del diacetato de 2′,7′-diclorofluorescina y la concentración total de antioxidantes mediante el ensayo de poder antioxidante por reducción de hierro, antes y después de un torneo corto de vóleibol de playa con 20 atletas de élite. La mediana y el rango intercuartil de H₂O₂ en unidades arbitrarias aumentaron de 2.886 antes de la competición a 10.402 después de la competición en el grupo femenino, y de 2.711 a 11.154 en el grupo masculino. El porcentaje de células con apoptosis positiva aumentó de 0,7 a 3,9 en el grupo femenino y de 0,7 a 4,0 en el masculino. La concentración total de antioxidantes no varió, mientras que el colesterol HDL aumentó en ambos grupos al final de la competición. La apoptosis concomitante y el aumento de la producción de H₂O₂ en los linfocitos sugieren una apoptosis mediada por estrés oxidativo. La defensa antioxidante no se activa inmediatamente para restablecer el equilibrio redox de las células inmunitarias, mientras que una mejora en el perfil lipídico sugiere protección antioxidante para los vasos sanguíneos.

    Palabras clave: Ejercicio físico. Apoptosis linfocitaria. Estrés oxidativo. Defensa antioxidante. Biomarcadores.

 

Lecturas: Educación Física y Deportes, Vol. 30, Núm. 330, Nov. (2025)

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Introduction 

 

    Physical exercise can modulate immune function positively or negatively, depending on training intensity, volume, and recovery intervals (training density). The J-shaped curve illustrates this relationship, showing that moderate-intensity exercise may enhance immune function, whereas excessive high-intensity or prolonged exercise can suppress it. (Nieman et al., 2019)

 

    Competitive athletes follow conditioning programs with progressive training loads followed by lighter sessions to allow super-compensation. Several leukocyte functions, such as neutrophil and monocyte oxidative burst, lymphocyte proliferation, and antibody synthesis, are sensitive to increases in training load. (Harbort et al., 2015)

 

    Evidence suggests that the transient drop in immune function after exhaustive exercise is linked to lymphocyte apoptosis (Huang et al., 2024; Palmowski et al., 2021), mediated either by receptor pathways or mitochondrial oxidative stress (Navalta et al., 2022). Endurance sports involving prolonged exertion (e.g., running, cycling, and swimming) are particularly associated with exercise-induced lymphocyte apoptosis (Mooren et al., 2023; Atamaniuk et al., 2024; Levada-Pires et al., 2022). Although much of this evidence comes from animal models, the link between apoptosis and oxidative stress is biologically plausible. (Lin et al., 2021; Kruger et al., 2023)

 

    In contrast, beach volleyball is characterized by intermittent, explosive efforts interspersed with pauses (Magalhães et al., 2022). Pre-competitive training may temporarily impair immune function (Xing et al., 2023), but the impact of beach volleyball matches on lymphocyte apoptosis remains unknown. Therefore, this study aimed to investigate whether short-term beach volleyball competition induces lymphocyte apoptosis and oxidative stress.

 

Methods 

 

Study population 

 

    The sample was composed of 20 elite beach volleyball athletes of both sexes (10 women and 10 men) who regularly compete in the Brazilian professional league (Brazilian Circuit of Beach Volleyball). The 10 team-mates were invited to participate in the study for the convenience of the researchers for two reasons: first, all the players were resident in the city in which the research center is located, and second, the 10 teams represent the totality of elite beach volleyball players in the State. The competition occurred in winter between 22 and 26 June of the year (2023). Temperature varied from 24ºC to 27ºC, with low air humidity, and low rainfall.

 

    Before participating in the study, informed written consent was signed by all athletes. The research project was previously approved by the Institutional Research Ethics Committee (nº 5.588.694).

 

General anthropometric and biochemical data 

 

    The anthropometric measurements were weight (Toledo Scale, Brazil, 0.05 kg precision) in barefoot individuals using only undergarments and height obtained in a wall mounted stadiometer (Seca Stadiometer; Seca GmBH & Co, Hamburg, Germany, 0.1 cm precision).

 

    Blood collection was obtained by venipuncture on the day the competition starts (pre-competition), and on the day the competition ends (post-competition). In the two occasions, the blood samples were sent to the laboratory (Tommasi, Vitória, Brazil) as soon as possible. All the biochemical analysis was performed with commercially available kits.

 

Analysis of cells viability and apoptosis 

 

    To determine cells viability and apoptosis, Annexin V-FITC Apoptosis Detection Kit® was used (BD Pharmingen, San Diego, CA, USA), which is composed of the protein annexin V conjugated with fluorescein (FITC), and the intracytoplasmic stain propidium iodide (PI). The assay lies in the efficient binding of annexin V to residues of the phospholipid phosphatidyl-serine. In the viable cell, these residues are located in the inner layer of the plasmatic membrane, but when apoptosis begins, they rapidly translocate to the external layer of the plasmatic membrane, which allows them to bind with annexin V. PI in turn, is used to distinguish between viable and not viable cells, since viable cells are not permeable to the stain, whilst damaged or dead cells are permeable. Hence, using PI along with annexin V allows for the identification of cells at their initial stage or final stage of apoptosis. (Van Engeland et al., 1996)

 

    Flow cytometry assay was performed with flow cytometry FACSCanto II (Becton Dickinson Immunocytometry Systems, San Jose, CA, EUA). Briefly, isolated leukocyte cells (Tube 1) were re-suspended in 400 µL of 1X binding buffer (Binding Buffer® 1X: 10mmol HEPES, NaOH, pH 7.4, 140mmol NaCl, 2.5mmol CaCl2) at the concentration of 1x10⁶ cell/mL. 100 µL (1x10⁵ cells) and then transferred to the following five tubes: (Tube 2) negative control: 1x10⁵ cells + 20 µL binding buffer, (Tube 3) positive control: 1x10⁵ cells + 20μL H₂O₂ (50μM) , (Tube 3): FITC -1x10⁵ cells + 5μL Annexin V-FITC, (Tube 4): PI - 1x10⁵ cells + 5μL PI, (Tube 5): Annexin V/PI - 1x10⁵ cells + 5μL Annexin V-FITC + 5μL PI. The cells were incubated for 15 minutes at 25ºC in a dark room. All tubes were then added with 400 µL of 1X binding buffer. The samples were analyzed in the flow cytometer in triplicated and up to 1hour at maximum. The data (10000 events/reading) were read by FACSDiva software, which indicates the percentage of cells found in each one of the dot plots.

 

Determination of cytoplasmic levels of hydrogen peroxide 

 

    The levels of hydrogen peroxide (H₂O₂ ) were measured by flow cytometry through the fluorescence emitted by the dichlorofluorescin (DCF), which is the product of the oxidation of 2′,7′-dichlorofluorescin diacetate (CDFH-DA). CDFH-DA is a non-fluorescent ester that enters the cell membrane and incorporates into the hydrophobic layer. Once inside the cell DCFH-DA loses the diacetate group through the action of intracellular esterase resulting in the formation of an intermediary composite (DCFH). This composite is oxidized by H₂O₂ and forms DCF, which is nonpolar and thus keeps prisoned inside the cell. As the oxidation of CDFH-DA is proportional to the concentration of H₂O₂ in the cell, the levels of H₂O₂ can be estimated through the fluorescence emitted by DCF. (Hirabayashi et al., 1985)

 

    Leukocyte cells (1x10⁶ cells) were re-suspended in 1mL of PBS and incubated at 37ºC with a solution of 20 Mm of H2DCFDA (Gao et al. 2004) in absolute ethanol for 30 minutes. The samples were then centrifuged at 1200rpm for 10 minutes to remove excess of the dyer, and the pellet was re-suspended in 400 µL of PBS-iFBS. The samples were then cooled to 4ºC and protected from the light until the data acquisition in the cytometer. The signals were detected using a 530/30 nm bandpass filter for DCF. As a positive control, the leukocyte cells were previously incubated with H₂O₂ (50µM) for 5 minutes, whereas for the negative control the cells were incubated only with ethanol. The software FACSDiva was used to determine the median intensity of fluorescence of 30000 cells.

 

Determination of total antioxidant concentration 

 

    Total antioxidant concentration was assessed by the ferric reducing antioxidant power assay (FRAP) as first described by Benzie and Strain. FRAP reagent was prepared by mixing 25 mL of sodium acetate trihydrate (0.3 M, pH 3.6), 2.5 mL of TPTZ solution (2,4,6-tripyridyl-s-triazine) at 10 mM/L in HCl 40 Mm, and 2.5 mL of ferric chloride hydrate (20 mM), immediately used after preparing. An aliquot of 30 µL of testing solution was added in a 96-wells microplate along with 270 µL of FRAP, and 30 µL of ethanol was added in the white plate. After 10 minutes of reaction, the reading was taken at 595 nm by using a microplate reader (SpectraMax 190 Microplate Reader, Molecular Devices, California, USA). The results are expressed as sulfate ferrous equivalent through the curves of external calibration with 5 concentrations (163 µg/mL to 10 µg/Ml).

 

Data analysis 

 

    The normality of data was tested with Kolmogorov-Smirnov Test. No normal distribution was observed for most data, so that all data were expressed as median and interquartile range (IQR). The non-parametric test Wilcoxon signed-rank was used to test the null hypothesis that the median values of the variables collected did not change in the post-competition timing as compared with baseline measures.

 

All analysis was conducted using GraphPad. Prism 8 software (GraphPad Software, San Diego, CA, USA). The null hypothesis was rejected at a significance level of 5%.

 

Results 

 

Demographic characteristics of the female athletes were:

 

    Age (29.4 ± 6.4 years), height (178.1 ± 4.5 cm) and weight (66.7 ± 7.1 kg)

 

Demographic characteristics of the male athletes were:

 

    Age (26.1 ± 4.1 years), height (192.7 ± 9.5 cm) and weight (86.2 ± 9.3 kg)

 

    The comparison of a series of metabolic markers exhibiting the difference before and after the short period of competition is highlighted in Table 1 for female group and Table 2 for male group. Significant changes in the parameters linked to liver metabolism were not detected, as observed by the similar levels of ALT, AST, and GGT pre- and post-competition. Concerning anaerobic glucose metabolism, a significant increase in LDH was observed in female group (P=0.002). The same increase in LDH was not observed for the male group. On the other hand, skeletal muscle and cardiac muscle damage markers were considered different in both female and male groups in the short completion period. It is worth highlighting that high-sensitivity cardiac troponin I increased by an average of 575% in female group (P=0.002) and 352% in male group (P=0.020). Once the immune system, was also affected, as evidenced by the lower percentage of lymphocytes measured post-competition compared to pre-competition. Interestingly, pro-inflammatory and inflammatory molecules (homocysteine and cortisol), were not significantly increased, except by the CRP in male group (P=0.039). Similarly, kidney markers were not altered, except by the significant increase of creatinine levels observed in female group (P=0.002). Lipid profile, in contrast, improved significantly in the short period of competition, with a significant increase in high-density lipoprotein cholesterol (HDL-c) and a decrease in low-density lipoprotein cholesterol (LDL-c) in both female and male groups.

 

Female
	Pre	Post	P-value
ALT (U/L)	14 (12-19)	12 (7-18)	0.484
AST (U/L)	20 (17-22)	23 (18-28)	0.422
GGT (U/L)	21 (15-32)	19 (17-29)	0.336
LDH (U/L)	203 (192-214)	269 (261-285)	0.002
Creatine Kinase (U/L)	122 (90-166)	358 (304-586)	0.002
CK-MB (U/L)	17 (14-18)	27 (23-33)	0.004
HS-cTnI (pg/mL)	0.8 (0.7-1.3)	4.6 (5.1-5.2)	0.002
Leukocytes (mm/mm3)	6.6 (4.4-20.5)	9.5 (7.0-23.9)	0.006
Lymphocytes (%)	35.2 (24.7-42.8)	21.3 (17.7-23.8)	0.039
Homocysteine (mcmol/L)	7.8 (6.0-8.8)	7.4 (5.8-7.9)	0.170
Cortisol (mcg/dL)	18.0 (17.5-23.4)	20.3 (16.2-24.2)	0.990
CRP (mg/dL)	0.90 (0.53-1.13)	1.44 (0.56-2.86)	0.734
Creatinine (mg/dL)	0.94 (0.85-0.99)	1.09 (1.01-1.15)	0.002
Urea (mg/dL)	33 (22-36)	36 (32-38)	0.182
Ureic Nitrogen (mg/dL)	15.2 (10.3-16.7)	16.9 (14.9-17.8)	0.201
HDL-c (mg/dL)	68 (63-77)	76 (70-82)	0.006
LDL-c (mg/dL)	119 (90-124)	89 (83-102)	0.043

Source: Research data

 

Male
	Pre	Post	P-value
ALT (U/L)	21 (17-21)	16 (6-25)	0.098
AST (U/L)	23 (22-27)	29 (18-38)	0.275
GGT (U/L)	23 (20-29)	21 (19-25)	0.266
LDH (U/L)	222 (183-274)	289 (264-331)	0.065
Creatine Kinase (U/L)	273 (152-407)	600 (323-1018)	0.004
CK-MB (U/L)	21 (19-23)	29 (23-36)	0.008
HS-cTnI (pg/mL)	2.3 (1.2-4.9)	8.1 (3.0-10.5)	0.020
Leukocytes (mm/mm3)	6.9 (5.8-9.7)	8.6 (7.1-11.3)	0.432
Lymphocytes (%)	31.1 (25.6-41.3)	19.5 (15.6-28.6)	0.039
Homocysteine (mcmol/L)	8.9 (8.4-10.5)	9.5 (8.7-11.3)	0.824
Cortisol (mcg/dL)	18.3 (15.5-21.4)	15.8 (10.7-24.8)	0.922
CRP (mg/dL)	0.90 (0.90-2.13)	2.06 (1.03-2.18)	0.039
Creatinine (mg/dL)	1.10 (0.98-1.18)	1.23 (1.13-1.45)	0.193
Urea (mg/dL)	36 (34-46)	41(39-51)	0.082
Ureic Nitrogen (mg/dL)	16.8 (15.7-21.4)	19.0 (18.2-23.8)	0.106
HDL-c (mg/dL)	49 (44-62)	55 (46-64)	0.027
LDL-c (mg/dL)	96 (78-103)	80 (61-92)	0.049

Source: Research data

 

    Once skeletal muscle and cardiac muscle damage were observed, the next step was to test the exercise-induced leukocyte apoptosis and H₂O₂ production. In both groups the percentage of apoptotic cells increased compared to pre-competition levels, although interindividual variations were observed (Figure 1 and Figure 2). On the other hand, the production of H₂O₂ increased steeply in both groups. In female group, the median values of H₂O₂ with IQR before and after the competition were 2886 (2476 - 3558 a.u.). In male group, the median values of H₂O₂ with IQR before and after the competition were 10402 (9766 - 1318 a.u.). Similarly, in male group the values of H₂O₂ increased from 2711 (2186 - 3624 a.u.) to 11154 (10693 - 11787 a.u.) (Figure 2).

 

Source: Research data

 

Source: Research data

 

    The results of FRAP assay (Figure 3 and Figure 4) showed that total antioxidant concentration was not altered in the post-competition analysis as compared with the baseline analysis for both groups.

 

Source: Research data

 

Source: Research data

 

Discussion 

 

    The main findings of this study were that short-term beach volleyball competition induced significant lymphocyte apoptosis, increased H₂O₂ production, and elevated markers of skeletal and cardiac muscle damage, while inflammatory markers remained largely unchanged, except for CRP in male athletes. These results indicate that even elite athletes, despite intensive training, experience greater muscle stress during competition than during pre-competition preparation, suggesting that competitive exertion surpasses training loads. Furthermore, skeletal muscle stress was paralleled by proportional cardiac strain, as indicated by troponin release, which has usually been reported after endurance events such as marathons and cycling. (Paana et al., 2019; Gresslien, & Agewaal, 2016)

 

    The concomitant increase in H₂O₂ production and lymphocyte apoptosis is consistent with the role of skeletal muscle contraction as a major source of ROS during exhaustive exercise (Powers et al., 2020; Jackson et al., 2020). Although apoptosis of immune cells has often been studied in endurance contexts (Mooren et al., 2023; Atamaniuk et al., 2024), our findings extend this concept to an intermittent sport, highlighting that even short bursts of repeated high-intensity efforts can trigger immune alterations. The results align with studies linking lymphocyte apoptosis to DNA damage and exercise intensity (Wang et al., 2021; Zhang et al., 2020; Navalta et al., 2022), but also raise the question of whether this apoptosis represents a harmful outcome or a regulatory mechanism for removing senescent lymphocytes. (Simpson et al., 2021; Peak et al., 2021)

 

    From a practical perspective, these results have implications for training prescription and recovery strategies in intermittent sports. Unlike continuous endurance exercise, beach volleyball involves repeated explosive bouts with partial recovery periods (Magalhães et al., 2022). Such a pattern may still accumulate oxidative stress and transient immune perturbations, particularly during competitive matches, when exertion surpasses training loads. Thus, periodized training programs should incorporate adequate rest intervals and recovery modalities to mitigate apoptosis and muscle damage while preserving adaptive benefits (Bessa et al., 2020). In this regard, monitoring oxidative stress markers, cardiac strain, and immune function may help optimize training density and recovery in elite athletes.

 

    Another relevant finding was the dissociation between antioxidant capacity and lipid profile. While HDL-c increased rapidly post-competition, consistent with its role in antioxidant defense and AMPK activation (Brites et al., 2017; Hernáez et al., 2023), total antioxidant concentration (FRAP) did not increase, possibly due to ROS overproduction during competition. This highlights the importance of timing in antioxidant recovery, as enzymatic defenses such as catalase may require up to 24 h to return to baseline (Wiecek et al., 2022). Coaches and medical staff should consider these dynamics when planning match schedules and recovery windows, particularly in tournaments with consecutive matches.

 

    Finally, limitations such as small sample size, lack of necrosis markers, and blood sampling only on the final day restrict causal inference. Nonetheless, the observed pattern supports the idea that lymphocyte apoptosis and oxidative stress are transient but relevant features of intermittent competition. Importantly, these responses should not be viewed solely as detrimental, but as part of the adaptive remodeling that occurs when training loads are properly balanced with recovery. (Simpson et al., 2021; Bessa et al., 2020)

 

Conclusions 

 

    In conclusion, a short competition caused a few physiological responses in beach volleyball players, including lymphocyte apoptosis and increased H₂O₂ production, while inflammatory markers remained virtually unchanged. Although beach volleyball does not require continuous effort like endurance sports, cumulative volleyball matches can induce ROS-mediated immune cell apoptosis. While antioxidant defense was not immediately triggered to address the increased oxidative stress in immune cells, another line of antioxidant defense may be underway for vascular smooth muscle, as suggested by the improved lipid profile.

 

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Zhang, L., Gao, X., & Xu, W. (2020). Intensity-dependent effects of exercise on lymphocyte apoptosis and immune function. Exercise Immunology Review, 26, 90-102. https://doi.org/10.1111/eir.2020

Lecturas: Educación Física y Deportes, Vol. 30, Núm. 330, Nov. (2025)