Repeated Moderate and Severe Heat Exposure Has Divergent Effects on Skeletal Muscle Mitochondrial, Proteostatic and Neuromuscular Outcomes in Mice

ABSTRACT Aim Repeated heat exposure may promote adaptive remodeling or cumulative tissue burden depending on thermal dose, but its graded molecular and functional consequences during recovery remain unclear. We examined whether repeated moderate (39°C) and severe (41°C) whole‐body hyperthermia produce distinct skeletal muscle trajectories involving mitochondrial dynamics, proteostasis, contractile gene programming, and motor behavior. Methods Adult male Swiss mice underwent 15‐min daily whole‐body heat exposure for 7 days at 39°C or 41°C core temperature and were assessed immediately or after 7 days of recovery. Motor and behavioral outcomes were evaluated using open‐field, elevated plus maze, grip strength, and rotarod tests. Gastrocnemius muscle was analyzed by qRT‐PCR, immunoblotting, and immunofluorescence for mitochondrial biogenesis, dynamics, mitophagy, proteostasis, and contractile phenotype markers. Cerebellar HSP60/HSP70 immunoreactivity and serum corticosterone were also assessed. Results At 39°C, recovery induced coordinated mitochondrial biogenesis and balanced fission‐fusion transcript upregulation. At 41°C, acute Ppargc1a induction ( p < 0.01) was followed by recovery‐phase mitophagy and oxidative‐response gene upregulation. Gastrocnemius ERK phosphorylation increased preferentially during 39°C exposure ( p < 0.05). HSP60 progressively declined across recovery groups, whereas HSP70 increased only after 41°C recovery ( p < 0.05). Grip‐related neuromuscular performance was impaired at 41°C in both phases ( p < 0.05), while rotarod performance remained intact. Cerebellar HSP70 was strongly suppressed by heat exposure ( p < 0.0001) and partially restored after 39°C recovery. Principal component analysis without group labels reproduced this divergence, yielding a dose × phase interaction axis ( p = 5.7 × 10 −7 ). Conclusion Moderate hyperthermia supports adaptive mitochondrial remodeling, whereas severe hyperthermia promotes stress‐related quality control, proteostatic strain, contractile suppression, and reduced grip‐related neuromuscular performance.