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This study investigated seasonal, sex and exercise-induced variations in the activity of key antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] in Shetland ponies. region (Pomeranian Voivodeship, northern part of Poland). Twenty-one healthy adult Shetland ponies (11 mares and 10 stallions) aged 6.5 ± 1.4 years were used in this study. All horses participated in recreational riding. The training session started at 10:00 am, lasted 1 hour and consisted of a cross-country ride including walking (5 min), trotting (15 min), walking (10 min), trotting (10 min), walking (5 min), galloping (5 min) and walking (10 min). Blood was collected from the animals’ jugular veins in the morning, 90 min after feeding, while the horses were in the stable (between 8.30 and 10 am) and immediately after the exercise test (between 11 am and 12 am). Blood samples were taken once per season for one year. The research highlights the complex interplay between environmental factors and physiological stressors in modulating antioxidant defence mechanisms. Significant seasonal variations in the activities of SOD, CAT and GPx were observed, with the enzymes showing increased sensitivity to exercise, particularly during the colder months. Mares showed a more pronounced exercise-induced decrease in SOD activity compared to stallions, especially during autumn and winter. Conversely, stable enzyme activity was observed in spring and summer, indicating reduced oxidative stress during milder seasons. Statistical analysis revealed significant seasonal differences in SOD and GPx activities, with higher coefficients of determination for SOD (R² = 0.45) compared to CAT and GPx. The study suggests that mares have a greater oxidative response to exercise in colder seasons, highlighting sex-specific differences in antioxidant defences. These findings contribute to the understanding of how seasonal and exercise-related stressors influence antioxidant enzyme activity and highlight the role of environmental adaptations in equine health and performance. The results have practical implications for optimising exercise regimes and antioxidant supplementation in equine management. Further research is needed to explore the underlying mechanisms of these differences and their wider implications for animal health.

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