Objective: To determine whether the amount of joint cartilage in healthy, middle aged men is stable or changes over time, and what factors may influence this. Methods: In a cohort study, 28 healthy men (70% of the original cohort; mean (SD) age, 51.9 (12.8) years) had baseline knee magnetic resonance imaging (MRI) of their dominant knee and repeat MRI of the same knee approximately 2.0 years later. Knee cartilage volume was measured at baseline and follow up. Risk factors assessed at baseline, including sex hormones and metabolic bone markers, were tested for their association with change in knee cartilage volume over time. Results: Mean (SD) reduction in tibial cartilage volume per year was 162 (93) μl. This represented a 2.8% reduction in total tibial articular cartilage per year (95% confidence interval, 0.2% to 5.5%). Tibial cartilage loss was associated with serum free testosterone level, independently of age, body mass index, baseline tibial cartilage volume tibial plateau area, and total bone mineral content. Overall, testosterone accounted for 14.5% (partial r 2) of the variation in change in tibial cartilage volume. There was a trend towards a positive association between tibial cartilage loss and urinary N-telopeptide cross-links of type I collagen (Ntx) (p = 0.057). Conclusions: Further studies will be required to determine whether hormonal manipulation or treatment with antiresorptive drugs will reduce the risk of knee osteoarthritis in men in later life.