Exercise and hypoxia independently stimulate free radical generation by human skeletal muscle (Bailey et al., 2007) subsequent to a decrease in intracellular oxygenation rather than an increase in oxygen (O₂) flux (Bailey et al., 2004). In the current study, we examined if the combination of exercise and inspiratory hypoxia would further compound regional tissue de-oxygenation and thus by consequence, increase oxidative stress. We also determined if inspiratory hyperoxia, by normalising tissue oxygenation, would subsequently attenuate oxidative stress. Eight healthy male volunteers aged 26 ± 2 (mean ± SD) were examined at rest in normoxia (21%O₂) and within 10 minutes of passive exposure to 12%O₂ (passive hypoxia) followed by a standardised cycling test to volitional exhaustion (active hypoxia). An additional measurement was performed following 5min passive recovery in 100%O₂ (passive hyperoxia). Absolute changes in oxy-haemoglobin concentration in cerebral (cO₂Hb) and skeletal (mO₂Hb) tissue were measured using continuous near-infrared spectroscopy (OXYMON Mk III). Differential path length factors of 6.0 and 4.0 were employed to correct for brain and muscle calculations. Plasma from a forearm antecubital vein was extracted into a high-sensitivity multi-bore aqueous cell prior to X-band electron paramagnetic resonance spectroscopy for the direct detection of the ascorbate free radical (A^‘-) expressed in arbitrary units (AU)/√line width in Gauss (G). All data were normally distributed (Shapiro-Wilk-W tests) and analysed using a one factor repeated measures ANOVA followed by Bonferroni-corrected paired samples t-tests. Compared to the normoxic control, the decrease in mO₂Hb observed during passive hypoxia (-1.3 ± 2.8 μmol/L, P < 0.05) was further compounded by exercise (-12.2 ± 5.3 μmol/L, P < 0.05) and increased markedly in hyperoxia (+9.6 ± 6.6 μmol/L, P < 0.05) Passive hypoxia decreased cO₂Hb (-4.5 ± 3.5 μmol/L, P < 0.05) with increases observed during exercise (+4.1 ± 10.7 μmol/L, P < 0.05) and hyperoxia (+23.1 ± 18.4 μmol/L, P < 0.05). Compared to the normoxic control, passive hypoxia increased plasma A^‘- by 150 ± 111 AU/√G, P < 0.05) with further increases observed during exercise (+371 ± 194 AU/√G, P < 0.05) and hyperoxia (+430 ± 164 AU/√G, P < 0.05). The present findings confirm the oxidative synergy that exists between hypoxia and exercise that may be related to the magnitude of muscle but not cerebral de-oxygenation. The hyperoxic data suggest that intracellular hyper-oxygenation may be equally as pro-oxidant as hypo-oxygenation.