To investigate the effects of heat stress (hyperthermia) on muscle degeneration- regeneration, the soleus muscles of adult male Wistar rats were injected bilaterally with a single injection of bupivacaine. The rats were assigned to a sedentary control (Con), heat stress (Heat), bupivacaine-injected (BPVC), or bupivacaine-injected plus heat stress (BPVC+Heat) group. Heat stress was induced in the Heat and BPVC+Heat groups by immersion of the lower half of the body into water maintained at 42 ± 1°C for 30 min 48 h after the injection of bupivacaine and every other day during the following 1 or 2 weeks. The soleus muscles in all groups were excised 24 h after the final bout of heat stress. Mean muscle weight, fiber cross-sectional area, myonuclear number, and heat shock protein 72 (Hsp72) and calcineurin (CaN) protein levels were lower in the BPVC than in the Con or Heat groups at both time points. In contrast, several of these parameters in the BPVC+Heat group were not different or higher than in the Con or Heat groups at the 1- and/or 2-week time points. The number of total and activated satellite cells, estimated by analyses of Pax7-, M-cadherin-, and MyoD+ nuclei, was greater in BPVC+Heat than all other groups. Combined the results indicate that heat stress-related activation of satellite cells and up-regulation of Hsp72 and CaN expression played an important role in the regeneration of the soleus fibers after bupivacaine injection.