Background Acute metabolic demands that promote excessive and/or prolonged reactive oxygen species production may stimulate changes in mitochondrial oxidative capacity. Purpose To assess changes in skeletal muscle H2O2 production, mitochondrial function, and expression of genes at the mRNA and protein levels regulating energy metabolism and mitochondrial dynamics following a hyperinsulinemic-euglycemic clamp in a cohort of 11 healthy premenopausal women. Methods Skeletal muscle biopsies of the vastus lateralis were taken at baseline and immediately following the conclusion of a hyperinsulinemic-euglycemic clamp. Mitochondrial production of H2O2 was quantified fluorometrically and mitochondrial oxidation supported by pyruvate, malate, and succinate (PMS) or palmitoyl carnitine and malate (PCM) was measured by high-resolution respirometry in permeabilized muscle fiber bundles. mRNA and protein levels were assessed by real time PCR and Western blotting. Results H2O2 emission increased following the clamp (P < 0.05). Coupled respiration (State 3) supported by PMS and the respiratory control ratio (index of mitochondrial coupling) for both PMS and PCM were lower following the clamp (P < 0.05). IRS1 mRNA decreased, whereas PGC1α and GLUT4 mRNA increased following the clamp (P ≤ 0.05). PGC1α, IRS1, and phosphorylated AKT protein levels were higher after the clamp compared to baseline (P < 0.05). Conclusions This study demonstrated that acute hyperinsulinemia induced H2O2 production and a concurrent decrease in coupling of mitochondrial respiration with ATP production in a cohort of healthy premenopausal women. Future studies should determine if this uncoupling ameliorates peripheral oxidative damage, and if this mechanism is impaired in diseases associated with chronic oxidative stress.