Maximal exercise in normoxia results in oxidative stress due to an increase in free radical production. However, the effect of a single bout of moderate aerobic exercise performed in either relative or absolute normobaric hypoxia on free radical production and lipid peroxidation remains unknown. To examine this, we randomly matched {according to their normobaric normoxic VO2peak [peak VO2 (oxygen uptake)]} and assigned 30 male subjects to a normoxia (n = 10), a hypoxia relative (n = 10) or a hypoxia absolute (n = 10) group. Each group was required to exercise on a cycle ergometer at 55% of VO2peak for 2 h double-blinded to either a normoxic or hypoxic condition [FiO2 (inspired fraction of O2) = 0.21 and 0.16 respectively]. ESR (electron spin resonance) spectroscopy in conjunction with ex vivo spin trapping was utilized for the direct detection of free radical species. The main findings show that moderate intensity exercise increased plasma-volume-corrected free radical and lipid hydroperoxide concentration (pooled rest compared with exercise data, P < 0.05); however, there were no selective differences between groups (statexgroup interaction, P > 0.05). The delta change in free radical concentration was moderately correlated with systemic VO2 (r2 = 0.48, P < 0.05). The hyperfine coupling constants recorded from the ESR spectra [aN = 13.8 Gauss, and a(H)beta = 1.9 Gauss; where 1 Gauss = 10(-4) T (telsa)] are suggestive of oxygen-centred free radical species formed via the decomposition of lipid hydroperoxides. Peripheral leucocyte and neutrophil cells and total CK (creatine kinase) activity all increased following sustained exercise (pooled rest compared with exercise data, P < 0.05), but no selective differences were observed between groups (state x group interaction, P > 0.05). We conclude that a single bout of moderate aerobic exercise increases secondary free radical species. There is also evidence of exercise-induced muscle damage, possibly caused by the increase in free radical generation.