The study of the physiological adaptations of skeletal muscle in response to eccentric (ECC) contraction is based on protocols in which exercise intensities are determined relative to the concentric (CON) reference exercise (as percentage of the CON maximal oxygen consumption, or VO_2max). In order to use similar exercise protocols in rats, we compared the VO₂ values during uphill (CON) and downhill (ECC) running tests. VO₂ was measured in 15 Wistar rats during incremental treadmill running exercises with different slopes: level (0%), positive (+15% incline: CON+15%) and negative (i15% incline: ECC-15%; and 130% incline: ECC-30%). Similar VO₂ values were obtained in the ECC-30% and CON+15% running conditions at the three target speeds (15, 25 and 35 cm/sec). Conversely, VO₂ values were lower (p < 0.05) in the ECC-15% than in the CON+15% condition (CON+15% VO₂/ECC-15% VO₂ ratios ranging from 1.86 to 2.05 at the three target speeds). Thus, doubling the downhill slope gradient in ECC condition leads to an oxygen consumption level that is not significantly different as in CON condition. These findings can be useful for designing animal research protocols to study the effects of ECC and CON exercise in ageing population or subjects suffering from cardiovascular diseases.