The aim was to investigate if acute recombinant human erythropoietin (rHuEPO) injection had an effect on mitochondrial function and if exercise would have an additive effect. Furthermore, to investigate if in vitro incubation with rHuEPO had an effect on muscle mitochondrial respiratory capacity. Eight healthy young men were recruited for this double-blinded randomized placebocontrolled crossover study. rHuEPO (400 IU/kg body wt) or saline injection was given intravenously, before an acute bout of exercise. Resting metabolic rate and fat oxidation were measured. Biopsies were obtained at baseline, 120 min after injection, and right after the acute exercise bout. Mitochondrial function (mitochondrial respiration and H2O2 emission) was measured in permeabilized skeletal muscle using high-resolution respirometry and fluorometry. Specific gene expression and enzyme activity were measured. Skeletal muscle mitochondrial respiratory capacity was measured with and without incubation with rHuEPO. Fat oxidation at rest increased after rHuEPO injection, but no difference was found in fat oxidation during exercise. Mitochondrial respiratory capacity was increased after rHuEPO injection when pyruvate was in the assay, which was not the case when saline was injected. No changes were seen in H2O2 emission after rHuEPO injection or acute exercise. Incubation of skeletal muscle fibers in vitro with rHuEPO increased mitochondrial respiratory capacity. Acute rHuEPO injection increased mitochondrial respiratory capacity when pyruvate was used in the assay. No statistical difference was found in H2O2 emission capacity, although a numerical increase was seen after rHuEPO injection. In vitro incubation of the skeletal muscle sample with rHuEPO increases mitochondrial respiratory capacity.