Many different disease states are characterized by mitochondrial dysfunction, which results in excessive reactive oxygen species (ROS) production. In contrast muscle contraction induces ROS generation suggesting there is an optimal range of ROS production necessary for proper cell function. It is unclear if ROS production is influenced by metabolic substrate flux as a result of the energetic demand of contraction. The purpose of this study was to determine the rate and source of ROS production in contracting single muscle fibers (SMF) cultured with different metabolic substrates. ROS production was assessed in SMF isolated from adult male mice exposed to different stimulation conditions and/or different sources of metabolic substrate. Mitochondrial membrane potential was also assessed in SMF under similar conditions. The results of this study demonstrate ROS generation is significantly influenced by metabolic substrate and larges increases in ROS do not affect mitochondrial membrane potential in intact SMF.