Hypoxia increases systemic vasodilation during exercise whereas breathing hyperoxia is known to increase systemic vascular resistance. Little is known about the effect of altered oxygen tensions on endothelial function, particularly following an acute bout of exercise. The purpose of this study was to investigate the effects of constant load cycling and different levels of inspired oxygen (FIO2) on endothelial function in healthy, recreationally active men. It was hypothesized that exercising while breathing normoxic or hypoxic gas would improve FMD, whereas hyperoxic gas would not alter FMD. Thirteen healthy, recreationally active males (22±3; mean±SD yrs) volunteered to participate. Subjects randomly completed three graded exercise tests breathing either 16% O2 (HYPO), 21% O2 (AIR) or 100% O2 (HYPER) to determine gas-specific maximal workload (Wmax) on separate days. Subjects then returned on three separate days and performed, 40-min, constant-load cycling trials at 50% of their gas specific Wmax. Baseline flow-mediated dilation (FMD), performed using standardized techniques, was measured at rest, after 30 min of gas exposure and 30 min following each exercise trial. Subjects remained on the test gas for the entire protocol. No differences in absolute FMD were observed for AIR (0.1±2.2 mm), HYPO (-0.7±1.2 mm) and HYPER (0.3±1.3 mm), relative FMD (1.2±4.5; -1.0±4.6; 1.1±3.3%, respectively), baseline diameter (-0.4±4.0; -0.2±2.3; 0.1±1.2mm, respectively), shear rate (19±133; -34±138; 26±120L.s-1, respectively) or time-to-peak dilation (16.9±31.2; -0.52±51; 7.6±24.7sec, respectively) were observed at rest or after exercise with any gas. These novel data show that in recreationally active males, neither FIO2 nor exercise have a significant effect on endothelial function at rest or following an acute bout of exercise. Vascular remodeling and alterations in antioxidant defense associated with training status in these individuals could account for the lack of change observed with FMD after exercise.