Abstract Anterior cruciate ligament (ACL) injuries are common in sports activities. The ideal loading conditions for a healing ligament are not known, but there is much evidence showing that load is important for maintaining normal ligaments. In this study we used a tissue culture model to investigate the changes in ligament due to loading and unloading. Tissue culture allows for excellent control of applied loads and it maintains cells in their extracellular matrix. The goal of this study was to examine the effects of cyclic loading on canine ACL in culture by measuring tissue stiffness, collagen biochemistry, and histology.