Objective: To investigate how running, cycling, and sedentary cardiovascular stress impact biomarkers of cartilage turnover acutely in subjects with knee osteoarthritis (OA). Design: This was a sequential, cross-over, clinical study. Forty subjects with primary knee OA underwent moderate-to-high-intensity cycling, running, and adrenaline infusion on separate days. Blood was sampled before, during, and at 6-time points after intervention. On a control day, similar samples were taken. Biomarkers of type II collagen degradation (C2M, T2CM, Coll2-1, Coll2-1NO2), formation (PRO-C2), and aggrecan degradation (ARGS) were measured. Results: Mean age was 60.4 years, 40% were male, 45% had cumulated Kellgren-Lawrence (KL)-grade (Right + Left knee) of 2 to 3 and 55% had 4 to 6. Analyzing overall changes, area under the curve was significantly lower compared with resting values for ARGS and C2M after cycling and for ARGS after running. Considering individual time points, peak changes in biomarker levels showed reduction in C2M shortly following cycling (T_20min = −12.3%, 95% confidence interval [CI]: −19.3% to −5.2%). PRO-C2 increased during cycling (T_10min = 14.0%, 95% CI = 4.1% to 23.8%) and running (T_20min = 16.5%, 95% CI = 4.3% to 28.6%). T2CM decreased after cycling (T_50min = −19.9%, 95% CI = −29.2% to −10.6%), running (T_50min = −22.8%, 95% CI = −32.1% to −13.5%), and infusion of adrenaline (peak, T_50min = −9.8%, 95% CI = −20.0% to 0.4%). A latent increase was seen in Coll2-1 240 minutes after running (T_260min = 21.7%, 95% CI = −1.6% to 45.1%). Conclusion: Exercise had an impact on cartilage markers, but it did not suggest any detrimental effect on cartilage. Changes following adrenaline infusion suggest a sympathomimetic influence on the serological composition of biomarkers.