Context Housing temperature is a critical regulator of mouse metabolism and thermoneutral housing can improve model translation to humans. However, the impact of housing temperature on the ability of wheel running exercise training to rescue the detrimental effect of diet-induced obese mice is currently not fully understood.Objective To investigate how housing temperature affects muscle metabolism in obese mice with regard to calcium handling and exercise training (ET) adaptations in skeletal muscle, and benefits of ET on adiposity and glucometabolic parameters.Methods Lean or obese female mice were housed at standard ambient temperature (22 degrees C) or thermoneutrality (30 degrees C) with/without access to running wheels. The metabolic phenotype was investigated using glucose tolerance tests, indirect calorimetry, and body composition. Molecular muscle adaptations were measured using immunoblotting, qPCR, and spectrophotometric/fluorescent assays.Results Obese female mice housed at 22 degrees C showed lower adiposity, lower circulating insulin levels, improved glucose tolerance, and elevated basal metabolic rate compared to 30 degrees C housing. Mice exposed to voluntary wheel running exhibited a larger fat loss and higher metabolic rate at 22 degrees C housing compared to thermoneutrality. In obese female mice, glucose tolerance improved after ET independent of housing temperature. Independent of diet and training, 22 degrees C housing increased skeletal muscle sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) activity. Additionally, housing at 22 degrees C elevated the induction of training-responsive muscle proteins in obese mice.Conclusion Our findings highlight that housing temperature significantly influences adiposity, insulin sensitivity, muscle physiology, and exercise adaptations in diet-induced obese female mice.