The aim of this study was to validate a non-invasive protocol to determine aerobic and anaerobic capacity of treadmill running rats. Thirteen male Wistar rats (90 days old) were submitted to 4 exercise tests, consisting of running at 25, 30, 35 and 40 m min-1, continuously until exhaustion. For the critical velocity (CV) and anaerobic running capacity (ARC) estimations, the hyperbolic curve (velocity versus time to exhaustion (tlim)) was linearized to V= CV+ARC/tlim, where the CV and ARC were linear and slope coefficients, respectively. In order to verify if the CV was the maximal aerobic intensity, the rats were submitted to the maximal lactate steady state test (MLSS) composed of three 25-minute tests of continuous running trials at 15, 20 and 25 m min-1, with blood collection every 5 minutes. The CV was obtained at 22.8±0.7 m min-1 and the ARC, at 26.80±2.77 m. The MLSS was observed at 20m min-1, with blood lactate 3.84 ± 0.31 mmol L-1. There was a progressive increase in lactate concentration at 25 m min-1. The CV and MLSS were different, but presented a high and significant correlation (r=0.81). These results indicate that the non-invasive protocol can be used for physical evaluation of aerobic running rats, but the ARC should still be further investigated.