Aim: Hypoxic exercise (HE) may have more pronounced effects on glucose homeostasis than exercise under normoxic conditions (NE), but effects on 24-h glucose profile and substrate utilization remain unclear. We investigated the effects of moderate-intensity HE compared to NE on 24-h glucose profile and substrate metabolism in overweight/obese individuals. Methods: Ten overweight/obese men with impaired glucose homeostasis participated in a randomized, single-blind, crossover trial. Participants performed moderate-intensity cycling exercise for 4 consecutive days under mild normobaric hypoxic (FiO2: 15%) or normoxic (FiO2: 21%) conditions at similar relative exercise intensity (2x30 min/d at 50% of maximal heart rate, with a ~4 week washout period. 24-h glucose levels and systemic oxygen saturation (SpO2) were monitored throughout the study. At day 5, plasma metabolites and substrate oxidation were determined during a mixed-meal test under normoxic conditions. Results: SpO2 and absolute workload were lower (both P<0.001), whilst heart rate was comparable during HE compared to NE. HE did not alter mean 24-h, daytime, and nighttime glucose concentrations, and measures of glycemic variability. However, the HE-induced decrease in SpO2 was positively correlated with HE-induced improvements in mean 24-h (rs=0.683, p=0.042) and daytime (rs =0.783, p=0.013) glucose concentrations. Conclusion: HE at similar relative exercise intensity reduces SpO2 and has comparable effects on mean 24-h glucose concentration and glycemic variability than NE in overweight/obese men with impaired glucose metabolism. Nevertheless, a more pronounced reduction in SpO2 during HE was associated with lower 24-h glucose concentrations, suggesting that a marked hypoxic stimulus is needed to improve glucose homeostasis.