Mechanical loading of articular cartilage can influence chondrocyte metabolism and lead to alterations in cartilage matrix composition. Most previous studies have focused on the effect of cyclic loading on cartilage mechanical properties and proteoglycan synthesis. However, the role of proteoglycans synthesized from cyclically loaded cartilage in response to an acute overload has not been elucidated. Therefore, we conducted studies where low intensity, intermittent cyclic loading was applied to chondral explants prior to an acute unconfined compression on the tissue. The chondral explants were randomly assigned to three groups: 7, 14, and 21 days of 10 cycles of 0.2 Hz sinusoidal loading at 0.5 MPa followed by an unloaded interval of 3,600 s. All explants were then taken to 25 MPa of unconfined compression. Biochemical assays were conducted to determine the tissue proteoglycan and hydroxyproline contents. The results showed cyclic preloading increased the proteoglycan content and mechanically stiffened the explants, making them more resistant to matrix damage and cell death under 25 MPa of unconfined compression up to 14 days. After 21 days of cyclic loading, however, the explants lost compressive stiffness and suffered more extensive damage in the unconfined compression test. This study investigated the role of cyclic loading in response of chondral explants to a potentially damaging, acute overload. In the long term, these types of studies may help understand the role of preconditioning of articular cartilage for in vitro or even in vivo studies of blunt force trauma to a joint.