The non-pharmacological treatment of osteoarthritis (OA) includes exercise therapy; however, little is known about the specific effect of exercise on the joint per se. The purpose of the present study was to investigate the direct effects of a load-bearing exercise upon cartilage in a single, human osteoarthritic joint determined by biochemical markers of cartilage turnover and inflammation in the synovial fluid (SF), serum and urine. Eleven subjects with OA of the knee(s), but with no other joint- or inflammatory disorders, volunteered for the study and had samples of blood, urine and synovial fluid drawn both at baseline and following 30-min one-legged knee-extension exercise. Workload: 60% of 1 RM (Repetition Maximum). Determination of cartilage oligomeric matrix protein (COMP), aggrecan, C-terminal collagen II peptide (CTX-II) and interleukin (IL)-6 were performed in synovial fluid (SF), serum and urine. A significant decrease was found in SF concentration of COMP following exercise, whereas aggrecan, CTX-II and IL-6 remained unchanged. No differences in any of the tested markers were found in serum and urine between baseline and post-exercise. Thirty minutes of mechanical loading of a single knee joint in human subjects with knee OA resulted in a reduced COMP concentration in SF.