The effects of heat stress on aquatic animals are increasingly being discerned, but little is known about the effects of heat stress on muscle meat quality or the flavor of muscle. This study aimed to evaluate the effects of heat stress on the muscle antioxidant properties, structural and physical properties (e.g., pH, muscle color, shear force, and expressible moisture), chemical composition (e.g., nucleotides, organic acids, amino acids, and minerals), and volatile substances of rainbow trout. We observed that the antioxidant capacity of muscle decreased after stress experiments at 22.5 °C for 24 h. The content of inflammatory factors notably increased (p < 0.05), the pH value and red value of muscle decreased (p < 0.05), the interfiber space increased, and several muscle fibers were broken. Heat stress changed the contents of nucleotides, organic acids, minerals, and amino acids in muscle. The contents of IMP and AMP, which play an important role in the flavor of muscle, decreased (p < 0.05). The contents of two amino acids that provide a sweet taste decreased; those of five amino acids that provide a bitter taste increased (p < 0.05). Heat stress also affected the amount and type of volatile substances in muscle, which affected muscle odor. These results suggest that heat stress may exert adverse effects on the oxidative stability, structure, meat quality, and flavor of muscle, requiring attention and prevention.