The mitochondrial consequences of reactive oxygen species (ROS) are commonly implicated in many acute and chronic diseases. Generally, ROS damage can be caused by various ROS-generating enzymes such as xanthine and NADPH oxidase, reverse electron transfer flow at complex I, and various environmental factors. ROS damage leads to a reduced mitochondrial function, via 1) cytochrome c release leading to mitophagy and apoptosis, 2) membrane damage leading to mitochondrial uncoupling, 3) cardiolipin peroxidation and 4) cristae remodelling. Studying the effect of ROS on mitochondrial function is complicated by the fact that the initial alteration in mitochondrial function is unknown, and a vicious cycle of damage-mediated ROS-damage is generally assumed. What the initial alterations in mitochondrial bio-energetic function are after acute ROS exposure is surprisingly little understood. Here, we determined the acute effects of pyrogallol, a superoxide (O2.-) and hydrogen peroxide (H2O2) donor on mitochondrial respiration in permeabilized skeletal muscle fibres. Methods Mitochondrial respiration of ~5 mg soleus muscle from 8 <1 year-old female Wistar rats was measured by a substrate-uncoupler-inhibitor-titration protocol using high-resolution respirometry (Oroboros, Austria). NADH-linked respiration was measured after the addition of malate, pyruvate and 5 mM ADP. Outer-mitochondrial membrane integrity was assessed by cytochrome c. Oxidative phosphorylation (OXPHOS) capacity was measured after additional succinate, and maximal uncoupled electron transport capacity by FCCP. Mitochondrial complex I was blocked by rotenone to assess succinate-linked respiration. Values after the addition of 0 (CON), 50 and 100 µM pyrogallol were compared by ANOVA. Since pyrogallol converts oxygen into O2.- and H2O2, we corrected for this increased background respiration. Results Mitochondrial leak respiration was significantly lower after pyrogallol exposure (both p<0.001), with the lowest values at the highest pyrogallol concentration. NADH-stimulated respiration was lower after exposure to 100 µM (16±9 pmol O2/s/mg; mean±SD) compared to 50 µM (49±13 pmol O2/s/mg) compared to CON (56±9 pmol O2/s/mg). Maximal OXPHOS only tended to be lower after 100 µM pyrogallol (p=0.07). Succinate-linked respiration was not different between groups (p=0.61), indicative of mitochondrial complex I dysfunction upon acute exposure to pyrogallol. This was confirmed by a lower normalised respiration for NADH-substrates after 50 µM pyrogallol (0.50±0.12) and 100 µM pyrogallol (0.31±0.15) versus CON (0.60±0.08, p=0.003). Normalised succinate-linked respiration was significantly higher after pyrogallol exposure (p=0.04). There was a dose-dependent effect on mitochondrial outer-mitochondrial membrane damage after pyrogallol exposure. Conclusion The acute exposure of skeletal muscle to O2.- and H2O2 resulted in complex I dysfunction and mitochondrial outer-membrane damage, possibly due to mitochondrial supercomplex instability. Future experiments are aimed to understand whether ADP-sensitivity is altered after pyrogallol exposure and if stabilizing cardiolipin with SS-31 can alleviate these negative effects.