Context: Anterior cruciate ligament reconstruction (ACLR) is a known risk factor for knee osteoarthritis (OA). Since no disease-modifying treatments for OA exist, it is critical to understand joint responses to physical activity following an ACLR. Understanding knee cartilage deformation through ultrasound may provide a better understanding of how knee cartilage responds to running, and how this may contribute to OA pathophysiology and risk. Objective: To compare medial femoral cartilage deformation, outcomes, and forces during running in females with and without a unilateral ACLR knee. Design: Cross-sectional laboratory study. Methods: Sixteen females (8 ACLR history, 8 controls) participated. ACLR participants had an average age of 20.75 (1.83) years, height of 165.07 (7.43) cm, mass of 65.97 (8.55) kg, and were 36.13 (18.74) months postsurgery; controls had an average age of 20.62 (1.84) years, height of 166.00 (5.76) cm, and mass of 63.18 (4.94) kg. Independent variables were group and limb, with dependent variables including patient reported outcomes Knee Injury and Osteoarthritis Outcome Score (KOOS), medial femoral cartilage thickness changes, and indirect measures of stance-averaged vertical ground reaction forces and vertical rate of loading normalized to body weight during treadmill running. Data were analyzed using nonparametric statistics with significance defined as P ≤ .05. Results: All participants exhibited cartilage thickness reduction after 30 minutes of running (P < .001), with no significant group or limb differences in cartilage deformation, vertical ground reaction forces, or vertical rate of loading. ACLR participants reported significantly poorer patient reported outcome scores across all KOOS subscales. Moderate negative correlations were found between involved cartilage percent change and KOOS Symptoms (P = .025) and Sport/Rec (P = .043). Conclusions: Cartilage thickness significantly decreased in all participants after running, with no group or limb differences observed despite lower patient-reported outcomes in the ACLR group. Exploring the relationship between KOOS scores and cartilage response to activity may inform future research and strategies to mitigate OA risk.