ABSTRACT: Importance:Persons with knee symptoms recognize the health benefits of engaging in physical activity, but uncertainty persists about whether regular strenuous physical activity or exercise can accelerate tissue damage. A sedentary lifestyle of inactivity or underloading may also be associated with deleterious joint health. Objective:To establish whether long-term strenuous physical activity participation and extensive sitting behavior are each associated with increased risk of developing radiographic knee osteoarthritis (KOA) in individuals at high risk for the disease. Design, Setting, and Participants:This cohort study analyzed data from the Osteoarthritis Initiative, a prospective longitudinal cohort study of men and women with or at an increased risk of developing symptomatic, radiographic KOA. Community-dwelling adults were recruited from 4 US sites (Baltimore, Maryland; Columbus, Ohio; Pittsburgh, Pennsylvania; and Pawtucket, Rhode Island) and were followed up for up to 10 years. Individuals were included if they had a baseline Kellgren and Lawrence grade of 0 in both knees and completed a PASE (Physical Activity Scale for the Elderly) questionnaire at baseline and at least 2 follow-up visits over an 8-year interval. Data analyses were conducted from May 2018 to November 2018. Exposures:Baseline to 8-year trajectories of strenuous physical activity participation and extensive sitting behavior were identified using group-based trajectory models. Main Outcomes and Measures:Incident radiographic KOA, defined as Kellgren and Lawrence grade 2 or higher in either knee by the 10-year follow-up visit. Results:A total of 1194 participants were included in the sample (697 women [58.4%]), with a baseline mean (SD) age of 58.4 (8.9) years and mean body mass index (BMI) of 26.8 (4.5). Four distinct trajectories of weekly hours spent in strenuous physical activities and 3 distinct trajectories of extensive sitting were identified. Long-term engagement in low-to-moderate physical activities (adjusted odds ratio [OR], 0.69; 95% CI, 0.48-1.01) or any strenuous physical activities (adjusted OR, 0.75; 95% CI, 0.53-1.07) was not associated with 10-year incident radiographic KOA. Persistent extensive sitting was not associated with incident KOA. Despite relatively mild symptoms and high function in this early-stage sample, 594 participants (49.7%) did not engage in any strenuous physical activities (ie, 0 h/wk) across 8 years, and 507 (42.5%) engaged in persistent moderate-to-high frequency of extensive sitting. Older age, higher BMI, more severe knee pain, non-college graduate educational level, weaker quadriceps, and depression were each associated with a persistent lack of engagement in strenuous physical activities. Conclusions and Relevance:Results from this study appeared to show no association between long-term strenuous physical activity participation and incident radiographic KOA. The findings raise the possibility of a protective association between incident KOA and a low-to-moderate level of strenuous physical activities.