["Acta Physiologica, EarlyView. ", "\nAbstract\n\nAim\nThe optimal exercise intensity to improve endothelial function remains unclear, as well as whether the addition of hypoxia could potentiate this function. Therefore, the aim of this study was to compare the effects of different exercise intensities in normoxia and hypoxia on vascular reactivity and nitric oxide (NO) bioavailability in mice.\n\n\nMethods\nC57BL/6 mice underwent treadmill running three times per week, for 4 weeks at either low, maximal or supramaximal intensity in normoxia or hypoxia (inspire oxygen fraction = 0.13). Vascular reactivity and expression of genes and proteins involved in NO production/bioavailability were assessed in aorta using isolated vessel tension experiments, RT‐qPCR and western blot, respectively. Circulating NO metabolites and pro‐/antioxidant markers were measured.\n\n\nResults\nHypoxic exercise improved both acetylcholine‐induced vasorelaxation and phenylephrine‐induced vasoconstriction compared to normoxic exercise, independently of intensity. In hypoxia, a higher acetylcholine‐induced vasorelaxation was observed with high intensities (supramaximal and maximal) compared to low intensity. Exercise protocols modulated endothelial nitric oxide synthase (eNOS) and α1‐adrenergic receptor (α1‐AR) mRNA level, but not superoxide dismutase 3 (SOD3) and p47phox. No significant differences were observed for protein expression of α1‐AR, total eNOS, phosphorylated eNOS, SOD isoforms and p47phox. However, plasma SOD and catalase activities were significantly increased in hypoxic supramaximal compared to hypoxic low intensity, while concentration of nitrotyrosine significantly decreased. The latter was also observed in hypoxic maximal and supramaximal compared to the same intensities in normoxia.\n\n\nConclusion\nHypoxic high‐intensity exercise increases NO bioavailability and improves vascular function, opening promising clinical perspectives for cardiovascular disease prevention.\n\n"]