Heat stress causes morbidity and mortality in both humans and animals and is a pressing concern as average global temperatures continue to rise due to global warming. Commercial animal agriculture is also compromised resulting in reductions in animal production parameters including carcass value, milk yield, reproduction, and fertility with economic losses estimated at $2.4 billion dollars annually in the United States. Although heat related illness poses a threat to human and animal health, welfare, and performance worldwide, mechanisms of a heat stress-induced injury are not well understood. We have previously found increased free radical injury following 12 and 24 hours of heat stress in oxidative, but not glycolytic skeletal muscle, which implicates mitochondria as contributors to cellular dysfunction. The objective of this study was to determine the extent to which heat stress altered mitochondrial dynamics in oxidative skeletal muscle. To address these objectives two studies were performed. In the first investigation crossbred gilts were subjected to thermoneutral (TN; 20 à °C) or heat stress (HS; 35 à °C) conditions for 1 or 3 days. Autophagic signaling and flux were found to be uncoupled by heat stress such that signaling was increased, however, flux was inhibited, causing autophagic stalling prior to lysosomal degradation. Furthermore, we found increased mitochondrial content, independent of biogenic signaling, indicating an accumulation of dysfunctional mitochondria. Previous work from our lab investigating heat-induced injury determined that 6 hours of heat stress in porcine skeletal muscle is an initial time point mediating heat-induced modifications. The purpose of our second study was to further evaluate the effects of heat-induced changes following 6 hours of stress in oxidative skeletal muscle. 24 male mice were randomly assigned to thermoneutral (TN; 21 à °C) or heat stress (HS; 37 à °C) conditions for six hours. Following treatment the animals were euthanized and the oxidative portion of gastrocnemius and soleus were recovered and divided for measurement of mitochondrial respiration, ROS production, metabolic flexibility, and autophagic signaling. Under these conditions heat stress did not cause mitochondrial dysfunction. Further, maintenance of mitochondrial function was independent from increased autophagic signaling and flux as these were similar between groups. These data demonstrate that although 6 hours of heat stress does not cause any changes, heat-induced injury is observed with longer periods of exposure leading to cellular and molecular dysfunction.