Treadmill Exercise Protocol for Mice

Mice were acclimatized to the motorized treadmill (Exer 3/6 Treadmill, Columbus Instruments, Columbus OU, USA) by running 30 min/day for 3 days at 5 m/min, 0° inclination. Exercise was performed as follows: 5 min of warmup at 5 m/min after which time the speed was increased 1 m/min every minute until the speed of 10 m/min. Then the mice ran 20 min at 10 m/min, 20 min at 11 m/min, and finally, 20 min at 12 m/min [51 (link)]. Mice subjected to physical preconditioning before HU ran for seven consecutive days. Animals of all experimental groups were fasted 2 h prior to the sacrifice. Mice were sacrificed by cervical dislocation, and gastrocnemius (Gas) muscles were dissected and immediately frozen in liquid nitrogen and stored at −80 °C.