The purpose was to assess the agreement in measures of acute knee cartilage thickness and composition change after loading in clinical knee osteoarthritis (OA) between two magnetic resonance imaging (MRI) acquisition approaches:​ (1) single sequence approach using quantitative double-echo in steady-state (qDESS), which allows simultaneous morphological and compositional scanning, versus (2) multi-sequence approach that captures morphology (fast spoiled gradient recalled (FSPGR) or qDESS) and composition (multi-echo spin echo (MESE)) separately. Twenty adults with clinical knee OA participated. 3T MR scans were acquired before and immediately after a 25-min treadmill walk at a standardized speed. Changes in knee cartilage thickness and T2 were assessed. Pre-activity, strong agreement was observed in cartilage thickness captured with qDESS and FSPGR (concordant correlation coefficients 0.842–0.935). Pre-activity, we observed greater absolute cartilage thickness with qDESS compared to FSPGR in femoral cartilage. From pre- to post-activity, qDESS showed change in cartilage thickness in the medial femur (−0.088 ± 0.11 mm, p = 0.002), lateral tibia (−0.042 ± 0.65 mm, p = 0.011) and trochlea (−0.027 ± 0.05 mm, p = 0.024);​ whereas FSPGR showed a change only in the lateral tibia (−0.064 ± 0.08 mm, p = 0.002). From pre- to post-activity, qDESS showed reduced T2 in all cartilage regions;​ whereas qDESS + MESE and FSPGR + MESE detected T2 changes in the patella (−1.90 ± 3.00 ms, p = 0.013, and −1.80 ± 2.18 ms, p = 0.002, respectively). qDESS detects transient changes in knee cartilage due to loading in clinical knee OA.