OBJECTIVE Walking exercise alleviates some symptoms, such as pain, in patients with mild to moderate knee osteoarthritis (OA). However, a major concern is that weightbearing exercise on osteoarthritic joints may exacerbate articular cartilage degradation. Loading of proteoglycan depleted articular cartilage in vitro increased expression of the chondroitin sulphate epitope 3B3, suggesting that loading may influence metabolism of osteoarthritic cartilage. This study aimed at evaluating the effects of walking exercise on articular cartilage metabolism in patients with knee OA, as reflected by changes in concentrations of synovial fluid markers. METHODS Thirty elderly patients with knee OA (Kellgren-Lawrence grades II to IV) were randomly allocated to control (n = 15) and 12 week exercise (n = 15) groups. Synovial fluid obtained from 21 of the patients at time zero and after 12 weeks was examined by enzyme linked immunosorbent assay (ELISA) for the chondroitin sulphate epitopes 3B3 and 7D4, and by a dye binding assay with 1, 9-dimethylmethylene blue for total sulphated glycosaminoglycan (GAG) concentrations. The 3B3/GAG and 7D4/GAG ratios were calculated. RESULTS No significant changes in concentrations of 3B3, 7D4, GAG, 3B3/GAG, or 7D4/GAG between time zero and 12 weeks were found in either group. However, there were significant declines in 3B3 (p=0. 001), GAG (p=0.007), and the 3B3/GAG ratio (p=0.049) with aging. CONCLUSIONS Twelve weeks of walking exercise had no demonstrable adverse effects on articular cartilage metabolism, as reflected by the concentrations of synovial fluid GAG or the chondroitin sulphate epitopes 3B3 and 7D4.