Introduction: Reactive oxygen and nitrogen species (RONS) are produced as a response to exercise and play a part in guiding the adaptive response to physical training. Exercise in the heat has been shown to further increase accumulation of RONS in systemic circulation; however, a high anti-oxidative response or heat acclimatization mechanisms could attenuate this response. In a previous study, we found reduced RONS concentrations in trained athletes following strenuous cycling in the heat. Therefore, this secondary analysis assessed whether these reduced RONS serum con-centrations are induced by an increased serum antioxidant capacity. Methods: Twelve male cyclists (V̇O2peak: 60 ± 4 ml ∙ kg-1 ∙ min-1) completed a 60-minute constant workload trial (55% peak power output, ambient temperature 30.4 ± 0.6°C) with and without ice vest in a randomized order. The core body temperature (Tcore) was measured by an ingestible capsule. Blood samples were col-lected before and after each trial to determine superoxide dismutase (SOD) and catalase (CAT) activity, total antioxidant capacity (TAC) and RONS. Due to the absence of between-group dif-ferences, data of both conditions were pooled. Results: Tcore statistically increased (p < 0.001) over the experimental trials (+6.0 ± 1.6%, effect size (ES) = 5.6). Concentrations of RONS (-17.2 ± 15.5%, p < 0.001, ES = 1.0) and TAC (-8.9 ± 22.9%, p = 0.04, ES = 0.7) statistically decreased, while the activity of CAT (+15.5 ± 84.0%, p = 0.90, ES = 0.04) and SOD (+9.2 ± 58.7%, p = 0.98, ES = 0.01) remained un-changed. Conclusions: Reduced serum RONS concentrations after strenuous cycling in the heat were not associated with upregulation of serum antioxidant capacity in trained athletes. The prooxidant-antioxidant balance may rather be regulated at a myocellular level and should be further assessed in future studies.