Significant evidence exists that trauma to a joint produced by a single impact load below that which causes subchondral bone fracture can result in permanent damage to the cartilage matrix, including surface fissures, loss of proteoglycan, and cell death. Limited information exists, however, on the effect of a varying impact stress on chondrocyte biophysiology and matrix integrity. Based on our previous work, we hypothesized that a stress-dependent response exists for both the chondrocyte’s metabolic activity and viability and the matrix’s hydration. This hypothesis was tested by impacting bovine cartilage explants with nominal stresses ranging from 0.5 to 65 MPa and measuring proteoglycan biosynthesis, cell viability, and water content immediately after impaction and 24 hours later. We found that proteoglycan biosynthesis decreased and water content increased with increasing impact stress. However, there appeared to be a critical threshold stress (15–20 MPa) that caused cell death and apparent rupture of the collagen fiber matrix at the time of impaction. We concluded that the cell death and collagen rupture are responsible for the observed alterations in the tissue’s metabolism and water content, respectively, although the exact mechanism causing this damage could not be determined.