Rest is a common treatment for overuse injuries, but its effectiveness on gene expression has not been systematically evaluated under controlled experimental conditions. We asked whether genes regulated in the supraspinatus tendon as a result of overuse would return to normal levels after 2 or 4 weeks of rest. We used a rat model of tendon overuse that generates reproducible changes in the histology, geometry, gene expression, and mechanical properties consistent with an overuse injury. Animals were subjected to the overuse protocol for 2 or 4 weeks followed by either 2 or 4 weeks of rest. Microarray analysis was used to measure global changes in gene expression after the overuse plus rest protocol. Genes upregulated as a result of the overuse returned to near normal levels after rest in most animals. The biochemical composition of the tendon was similar to normal after the imposed rest period, except for slightly lower collagen content. These results suggest as little as 2 weeks of rest is often sufficient to recover from the molecular and biochemical effects of 2 and 4 weeks of overuse in this rat model.