Background: Patients with anterior cruciate ligament reconstruction (ACLR) have demonstrated morphological and compositional changes in femoral articular cartilage. However, it is unclear how running biomechanics are associated with femoral cartilage thickness and composition for both ACLR patients and controls. Objectives: (1) to compare measures of femoral cartilage thickness and composition between ACLR patients and matched non-ACLR controls at resting, (2) to investigate how 30 minutes of running influences the aforementioned measures for ACLR patients and controls, and (3) to investigate relationships between running biomechanics and knee cartilage thickness and composition in ACLR patients and controls. Methods: Twenty ACLR patients (age: 23 ± 3 years; mass: 69.7 ± 9.9 kg; time post ACLR: 14.6 ± 6.1 months) and 20 matched non-ACLR controls (age: 22 ± 2 years; mass: 67.1 ± 10.9 kg) participated in the study. A running session required both groups to run for 30 minutes at a self-selected speed. Before and after running we measured femoral cartilage thickness via ultrasound imaging. An MRI session consisted of T2 mapping. Independent t-tests were used to examine differences in femoral cartilage thickness and T2 relaxation time at resting, and thickness changes following the run between the two groups. Pearson correlations were used to explore relationships between running biomechanics and femoral cartilage thickness and relaxation time at resting. Results: The ACLR group showed longer T2 relaxation times in three regions of the medial femoral condyle at resting compared with the control group (overall: 54.9 ± 14.2 vs. 39.3 ± 8.2 ms, P = 0.001; central: 51.2 ± 16.6 vs. 34.9 ± 13.2 ms, P = 0.006; posterior: 50.2 ± 10.1 vs. 39.8 ± 7.4 ms, P = 0.006). Following the run, the ACLR group showed greater deformation in the medial femoral cartilage than the control group (0.03 ± 0.01 vs. 0.01 ± 0.01 cm, P = 0.001). Additionally, the ACLR group showed significant negative correlations between resting T2 relaxation time in the central region of the medial femoral condyle and peak vGRF, and vertical impulse (r = -0.53, P = 0.013; r = -0.46, P = 0.041, respectively) during running. Conclusions: The ACLR group showed greater water content in medial femoral cartilage and greater deformation in medial femoral cartilage thickness following 30 minutes of running compared with the controls. In addition, the ACLR group demonstrated significant negative correlations between water content in medial femoral cartilage and vGRF. Our findings suggest that those who are at least 24 months post-ACLR have degraded cartilage composition and their cartilage is more sensitive to joint loading morphologically.