Aerobic training can improve vascular endothelial function in-vivo. The aim of this study was to elucidate the mechanisms underlying this improvement in isolated human microvascular endothelial cells. Sedentary males, aged 57 ± 6 years completed 8 weeks of intense aerobic training. Resting muscle biopsies were obtained from the thigh muscle and used for isolation of endothelial cells (pre n = 23, post n = 16). The cells were analyzed for mitochondrial respiration, H₂O₂ emission, glycolysis, protein levels of antioxidants, NADPH oxidase, endothelial nitric oxide (NO) synthase and prostacyclin synthase (PGI2S). In-vivo microvascular function, assessed by acetylcholine infusion and arterial blood pressure were also determined. Endothelial mitochondrial respiration and H₂O₂ formation were similar before and after training whereas the expression of superoxide dismutase and the expression of glutathione peroxidase were 2.4-fold (p = 0.012) and 2.3-fold (p = 0.006) higher, respectively, after training. In-vivo microvascular function was increased by 1.4-fold (p = 0.036) in parallel with a 2.1-fold increase in endothelial PGI₂S expression (p = 0.041). Endothelial cell glycolysis was reduced after training, as indicated by a 65% lower basal production of lactate (p = 0.003) and a 30% lower expression of phosphofructokinase (p = 0.011). Subdivision of the participants according to blood pressure at base-line (n = 23), revealed a 2-fold higher (p = 0.049) rate of H₂O₂ production in endothelial cells from hypertensive participants. Our data show that exercise training increases skeletal muscle microvascular endothelial cell metabolism, antioxidant capacity and the capacity to form prostacyclin. Moreover, elevated blood pressure is associated with increased endothelial mitochondrial ROS formation.