Autophagy is a major intracellular degradation process that is essential for the clearance of unnecessary proteins/organelles and the maintenance of cellular homeostasis. The inhibition of autophagy results in cellular consequences associated with many skeletal muscle pathologies, and therapies designed to elevate autophagic activity may provide protection from such pathologies. Acute exposure to low levels of heat has therapeutic effects; however, the impact of heat on skeletal muscle autophagy remains unclear. In the present study, C2C12 myotubes were maintained at 37°C thermoneutral (TN) or heated at 40°C heat treatment (HT) for 1 h. Myotubes were harvested immediately after heating, or returned to 37°C for recovery of 2 or 24 h. HT resulted in an elevation in pAMPK (T172), Beclin-1, and LC3 II, a marker for autophagosome formation, but no change in p62. In the context of autophagy inhibition with Bafilomycin A1, HT resulted in lower LC3 II compared to TN. The applied heat load induced the heat shock response, as evidenced by immediate upregulation of HSF1 and Hsp70. Hsp70 continued to increase during recovery, whereas pHsp27 was downregulated acutely in response to HT, but retuned to TN levels by 2 h of recovery. HT also reduced the phosphorylation of the MAP-kinases p38 and JNK. These findings suggest that an acute, short bout of mild heat may be beneficial to skeletal muscle by increasing AMPK activity, markers of autophagasome formation, and the heat shock response.