As global temperature shifts due to anthropogenic impacts, the frequency and magnitude of extreme temperatures in shallow aquatic ecosystems are expected to increase. Previous studies performed on fish under fluctuating temperature regimes have reported increased physiological stresses, compared to stable thermal conditions, leading to morphological, behavioral and biochemical adaptations. Moreover, many studies have reported the effects of warmer temperatures on numerous aquatic species, but seasonal temperature changes experienced in aquatic systems are understudied. We surveyed changes in the bioenergetic machinery of a native fish population ( Lepomis macrochirus) in the temperate zone of North America and observed season-dependent thresholds in the thermal sensitivity of the oxidative phosphorylation system. Liver mitochondria from L. macrochirus show significantly higher uncoupled proton conductance and cytochrome c oxidase (COX) activity and reduced respiratory control ratios in individuals captured in the fall compared to specimens collected during summer and spring. Somatic indices, such as relative weights and hepatosomatic indices also showed significant differences among seasons and critical thermal maxima decreased during the fall season. These findings suggest that mechanisms regulating proton conductance and COX activity are modulating mitochondrial function across seasons, to sustain physiological fitness in ectotherms inhabiting shallow, inland aquatic habitats.