Hyperthermia therapy has recently emerged as a clinical modality used to finely tune heat stress inside the human body for various biomedical applications. Nevertheless, little is known regarding the optimal timing or temperature of heat stress that is needed to achieve favorable results following hyperthermia therapy for muscle regeneration purposes. The regeneration of skeletal muscle after injury is a highly complex and coordinated process that involves a multitude of cellular mechanisms. The main objective of this study was to characterize the effects of hyperthermal therapy on the overall behavior of myoblasts during myogenic differentiation. Various cellular processes, including myogenesis, myofibrillogenesis, hypertrophy/atrophy, and mitochondrial biogenesis, were studied using systematic cellular, morphological, and pathway-focused high-throughput gene expression profiling analyses. We found that C2C12 myoblasts exhibited distinctive time and temperature-dependence in biosynthesis and regulatory events during myogenic differentiation. Specifically, we for the first time observed that moderate hyperthermia at 39°C favored the growth of sarcomere in myofibrils at the late stage of myogenesis, showing universal up-regulation of characteristic myofibril proteins. Characteristic myofibrillogenesis genes, including heavy polypeptide 1 myosin, heavy polypeptide 2 myosin, alpha 1 actin, nebulin and titin, were all significantly upregulated (p<0.01) after C2C12 cells differentiated at 39°C over 5 days compared with the control cells cultured at 37°C. Furthermore, moderate hyperthermia enhanced myogenic differentiation, with nucleus densities per myotube showing 2.2-fold, 1.9-fold and 1.6-fold increases when C2C12 cells underwent myogenic differentiation at 39°C over 24 hours, 48 hours and 72 hours, respectively, as compared to the myotubes that were not exposed to heat stress. Yet, atrophy genes were sensitive even to moderate hyperthermia, indicating that strictly controlled heat stress is required to minimize the development of atrophy in myotubes. In addition, mitochondrial biogenesis was enhanced following thermal induction of myoblasts, suggesting a subsequent shift toward anabolic demand requirements for energy production. This study offers a new perspective to understand and utilize the time and temperature-sensitive effects of hyperthermal therapy on muscle regeneration.