STUDY DESIGN: Controlled laboratory study. OBJECTIVES: To investigate the effects of instrument-assisted cross-fiber massage (IACFM) on tissue-level healing of knee medial collateral ligament (MCL) injuries. BACKGROUND: Ligament injuries are common and significant clinical problems for which there are few established interventions. IACFM represents an intervention that may mediate tissue-level healing following ligament injury. METHODS: Bilateral knee MCL injuries were created in 51 rodents, while 7 rodents were maintained as ligament-intact, control animals. IACFM was commenced 1 week following injury and introduced 3 sessions per week for 1 minute per session. IACFM was introduced unilaterally (IACFM-treated), with the contralateral, injured MCL serving as an internal control (nontreated). Thirty-one injured animals received 9 ACFM treatments, while the remaining 20 injured animals received 30 treatments. Ligament biomechanical properties and morphology were assessed at either 4 or 12 weeks postinjury. RESULTS: IACFM-treated ligaments were 43.1% stronger (P<.05), 39.7% stiffer (P<.01), and could absorb 57.1% more energy before failure (P<.05) than contralateral, injured, nontreated ligaments at 4 weeks postinjury. On histological and scanning electron microscopy assessment, IACFM-treated ligaments appeared to have improved collagen fiber bundle formation and orientation within the scar region than nontreated ligaments. There were minimal differences between IACFM-treated and contralateral, nontreated ligaments at 12 weeks postinjury, although IACFM-treated ligaments were 15.4% stiffer (P<.05). CONCLUSION: IACFM-accelerated ligament healing, possibly via favorable effects on collagen formation and organization, but had minimal effect on the final outcome of healing. These findings are clinically interesting, as there are few established interventions for ligament injuries, and IACFM is a simple and practical therapy technique. J Orthop Sports Phys Ther 2009;39(7):506-514, Epub 24 February 2009. doi:10.2519/jospt.2009.2997.