ObjectiveTo investigate the effect of unilateral anterior cruciate ligament (ACL) injury on cartilage thickness and composition, specifically laminar transverse relaxation time (T2) by magnetic resonance imaging (MRI), in younger and older participants and to compare within-person side differences in these parameters between ACL-injured and healthy controls. DesignQuantitative double-echo steady-state (qDESS) 3 Tesla MRI-sequences were acquired in both knees of 85 participants in four groups: 20–30 years: healthy, HEA20–30, n=24; ACL-injured, ACL20–30, n=23; 40–60 years: healthy, HEA40–60, n=24; ACL-injured, ACL40–60, n=14 (ACL injury 2–10 years prior to study inclusion). Weight-bearing femorotibial cartilages were manually segmented; cartilage T2 and thickness were computed using custom software. Mean and side difference in subregional cartilage thickness, superficial and deep cartilage T2 were compared within and between groups using non-parametric statistics. ResultsCartilage thickness did not differ within or between groups. Only the side difference in medial femorotibial cartilage thickness was greater in ACL20–30 than in HEA20–30. Deep zone T2 was longer in the ACL-injured than in the contralateral uninjured knees and than in healthy controls, especially in the lateral compartment. Most ACL-injured participants had side differences in femorotibial deep zone T2 above the threshold derived from controls. ConclusionIn the ACL-injured knee, early compositional differences in femorotibial cartilage (T2) appear to occur in the deep zone and precede cartilage thickness loss. These results suggest that monitoring laminar T2 after ACL injury may be useful in the diagnosing and monitoring early articular cartilage changes.