Key points: We determined if bed rest increased mitochondrially derived reactive oxygen species and cellular redox stress, contributing to the induction of insulin resistance. Bed rest decreased maximal and submaximal ADP-stimulated mitochondrial respiration. Bed rest did not alter mitochondrial H 2O 2 emission in the presence of ADP concentrations indicative of resting muscle, the ratio of H 2O 2 emission to mitochondrial O 2 consumption or markers of oxidative stress The present data suggest strongly that mitochondrial H 2O 2 does not contribute to bed rest-induced insulin resistance. Abstract: Mitochondrial H 2O 2 has been causally linked to diet-induced insulin resistance, although it remains unclear if muscle disuse similarly increases mitochondrial H 2O 2. Therefore, we investigated the potential that an increase in skeletal muscle mitochondrial H 2O 2 emission, potentially as a result of decreased ADP sensitivity, contributes to cellular redox stress and the induction of insulin resistance during short-term bed rest in 20 healthy males. Bed rest led to a decline in glucose infusion rate during a hyperinsulinaemic-euglycaemic clamp (−42 ± 2%; P < 0.001), and in permeabilized skeletal muscle fibres it decreased OXPHOS protein content (−16 ± 8%) and mitochondrial respiration across a range of ADP concentrations (−13 ± 5%). While bed rest tended to increase maximal mitochondrial H 2O 2 emission rates (P = 0.053), H 2O 2 emission in the presence of ADP concentrations indicative of resting muscle, the ratio of H 2O 2 emission to mitochondrial O 2 consumption, and markers of oxidative stress were not altered following bed rest. Altogether, while bed rest impairs mitochondrial ADP-stimulated respiration, an increase in mitochondrial H 2O 2 emission does not contribute to the induction of insulin resistance following short-term bed rest.