Treadmill Exhaustion Test for VO2max in Mice

Following one week of rest from acclimation training, mice were placed on the treadmill at 0° incline and the shock grid was activated. The treadmill speeds were then increased until exhaustion as follows: (speed, duration, grade)—(0 m/min, 3 min, 0°), (6 m/min, 2 min, 0°), (9 m/min, 2 minutes, 5°), (12m/min, 2 min, 10°), (15m/min, 2 min, 15°), (18, 21, 23, 24 m/min, 1 min, 15°), and (+1 m/min, each 1 min thereafter, 15°). Exhaustion (endpoint for treadmill cessation) was defined as the point at which mice maintained continuous contact with the shock grid for 5 seconds. Continuous contact is defined as any portion of the animal’s body coming in contact with the shock grid for a total of 5 seconds. During the test, occasional (~1–5 times per single animal test) 1–2 second tail contacts were observed when animals misstepped or were slow to response in the increase in intensity. VO_2max was determined by the peak oxygen consumption reached during this test when RER was >1.0. Maximum running speed was defined as the treadmill speed at which VO_2max was achieved (Table 1). All animals (within-subjects design, GXT_m and PXT_m) underwent pre- and post-test lactate assays (Lactate assay) one hour prior to and immediately following exercise testing).