1. The purpose of the present study was to verify whether a downhill running protocol was able to induce non-functional overreaching in > 75% of mice. 2. Mice were divided into control (C), trained (TR) and overtrained (OTR) groups. Bodyweight and food intake were recorded weekly. The incremental load test (ILT) and the exhaustive test (ET) were used to measure performance before and after aerobic training and overtraining protocols. 3. Although the bodyweight of the OTR group was lower than that of the C group at the end of Week 7, the food intake of the OTR group was higher than that of the C and TR groups at the end of Week 8. Evaluation of results from the ILT and ET revealed significant intra- and inter-group differences: whereas the parameters measured by both tests increased significantly in the TR group, they were significantly decreased in the OTR group. 4. In conclusion, this new overtraining protocol based on downhill running sessions induced non-functional overreaching in 100% of mice. © 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Wiley Publishing Asia Pty Ltd.