Acclimatization and Incremental Load Test (ILT)

As previously described (Pereira et al., 2012), the NT and OT groups were first acclimated to treadmill running (ZH-PT, Zhenghua, Anhui, China) at 10 m/min with 0% inclination for 10 min per day for a week (includes 5-day of treadmill adaptation, 1-day of ILT test, and 1-day of rest). Then, rodents performed the ILT with an initial intensity of 6 m/min at 0% inclination with 3 m/min increments every 3 min until exhaustion, which was defined as a mouse touching the end of the treadmill five times in 1 min. The mice were encouraged using physical prodding, and if a mouse became exhausted without completing the stage, the exhaustion velocity (EV = V + (n/b)*a, with V being the completed maximum speed, n is the duration in the incomplete stage, b being the duration of the stage, and a being the test increment) was calculated (Kuipers et al., 1985). The EV of each mouse was used to determine the intensity of the NT and OT protocols.