Tendons adapt to different mechanical stimuli through a remodeling process involving metalloproteinases (MMPs) and collagen synthesis. The purpose of this study was to investigate the activities of MMP-2 and MMP-9 and the collagen content in tendons after exhaustive acute exercise sessions over the course of 1, 3, or 6 days, with 1-hr or 3-hr rest periods between each session. Wistar rats were grouped into control (C), trained with 1-hr (groups 1d1h, 3d1h, and 6d1h) and trained with 3-hr (groups 1d3h, 3d3h and 6d3h) groups with rest periods between the treadmill running sessions, for 1, 3, and 6 days. The analysis of MMP-2 showed a larger presence of the latent isoform in the 1d3h group and a larger presence of the active isoform in the 6d3h group compared to the control. No differences were detected for MMP-9. A lower concentration of hydroxyproline was found in the 6d3h group compared to the 6d1h group. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed more prominent collagen bands in the 6d3h group, which was confirmed by Western blotting for collagen type I. A higher concentration of glycosaminoglycans was observed in the 3d3h group compared to the 3d1h group, and the 6d3h group presented the highest value for noncollagenous proteins compared to other groups. In conclusion, different rest periods between exercise sessions had different effects on the composition of the calcaneal tendon because a greater activation of MMP-2 and a reduction of total collagen were observed on day 6 of exercise with 3-hr rest periods compared to 1-hr rest periods. Anat Rec, 297:281-288, 2014. V C 2013 Wiley Periodicals, Inc.