Large temperature difference is reported to be a risk factor for human health. However, little evidence has reported the effects of temperature fluctuation on sarcopenia, a senile disease characterized by loss of muscle mass and function. Here, we demonstrate that higher diurnal temperature range in humans has a positive correlation with the prevalence of sarcopenia. Fluctuated temperature exposure (10–25 °C) accelerates muscle atrophy and dampens exercise performance in mid-aged male mice. Interestingly, fluctuated temperature alters the microbiota composition with increased levels ofParabacteroides_distasonis,Duncaniella_dubosiiand decreased levels ofCandidatus_Amulumruptor,Roseburia,Eubacterium. Transplantation of fluctuated temperature-shaped microbiota replays the adverse effects on muscle function. Mechanically, we find that altered microbiota increases circulating aminoadipic acid, a lysine degradation product. Aminoadipic acid damages mitochondrial function through inhibiting mitophagy in vitro. AndEubacteriumsupplementation alleviates muscle atrophy and dysfunction induced by fluctuated temperature. Our results uncover the detrimental impact of fluctuated temperature on muscle function and provide a new clue for gut-muscle axis.