IntroductionDiabetes mellitus (DM) impairs wound healing. The aim was to determine whether DM influences mitochondrial respiration in wounded skin (WS) and non‐wounded skin (NWS), in a pre‐clinical wound healing model of streptozotocin (STZ)‐induced diabetes.MethodsSix weeks after diabetes induction, two wounds were created in the back of C57BL/J6 mice. Using high‐resolution respirometry (HRR), oxygen flux was measured, in WS and NWS, using two substrate‐uncoupler‐inhibitor titration protocols, at baseline (day 0), day 3 and 10 post‐wounding, in STZ‐DM and non‐diabetic (NDM) mice. Flux control ratios for the oxidative phosphorylation (OXPHOS) capacity were calculated.ResultsA significant increase in mitochondrial respiration was observed in STZ‐DM skin compared to control skin at baseline. The OXPHOS capacity was decreased in WS under diabetes at day 3 post‐wounding (inflammation phase). However, at day 10 post‐wounding (remodeling phase), the OXPHOS capacity was higher in WS from STZ‐DM compared to NDM mice, and compared to NWS from STZ‐DM mice. A significant relative contribution of pyruvate, malate and glutamate (PMG) oxidation to the OXPHOS capacity was observed in WS compared to NWS from STZ‐DM mice, at day 10, while the relative contribution of fatty acid oxidation to the OXPHOS capacity was higher in NWS. The OXPHOS capacity is altered in WS from STZ‐DM compared to NDM mice across the healing process, and so is the substrate contribution in WS and NWS from STZ‐DM mice, at each time point.ConclusionHRR may be a sensitive tool to evaluate the underlying mechanisms of tissue repair during wound healing.