BACKGROUND: To optimize sampling and to understand sources of variation in biomarkers for osteoarthritis (OA), we evaluated variation due to activity and food consumption. METHODS: Twenty participants, with radiographic knee OA, provided serial serum and urine samples at 4 time points: before arising in the morning; after 1 h of light activity; 1 h after eating breakfast; and in the evening. Five serum (s) and 2 urinary (u) analytes were measured: hyaluronan (sHA); cartilage oligomeric matrix protein (sCOMP); keratan sulfate (sKS-5D4); transforming growth factor beta (sTGF-ss1); and collagen II-related epitopes (sCPII, uCTXII, and uC2C). Activity was monitored by an accelerometer. RESULTS: All serum biomarkers increased and one of the urinary biomarkers decreased after 1 h of non-exertional activity. Food consumption following activity was associated with a return of biomarker concentrations to baseline levels. Accelerometers proved to be a novel way to monitor protocol compliance and demonstrated a positive association between the mean level of activity and sCOMP concentration. Urinary CTXII varied the least but demonstrated both true circadian variation (peak in the morning and nadir in the evening) and the most robust correlation with radiographic knee OA. CONCLUSIONS: We confirm activity related variation in these markers. These data suggested that biomarkers also varied due to upright posture, glomerular filtration rate stimulated by food intake, and circadian rhythm in the case of uCTXII.