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Abnormal loading is thought to play a key role in the disease progression of cartilage, but our understanding of how cartilage compositional measurements respond to acute compressive loading in-vivo is limited. Ten healthy subjects were scanned at two timepoints (7 ± 3 days apart) with a 3 T magnetic resonance imaging (MRI) scanner. Scanning sessions included T1ρ and T2* acquisitions of each knee in two conditions: unloaded (traditional MRI setup) and loaded in compression at 40 % bodyweight as applied by an MRI-compatible loading device. T1ρ and T2* parameters were quantified for contacting cartilage (tibial and femoral) and non-contacting cartilage (posterior femoral condyle) regions. Significant effects of load were found in contacting regions for both T1ρ and T2*. The effect of load (loaded minus unloaded) in femoral contacting regions ranged from 4.1 to 6.9 ms for T1ρ, and 3.5 to 13.7 ms for T2*, whereas tibial contacting regions ranged from -5.6 to -1.7 ms for T1ρ, and -2.1 to 0.7 ms for T2*. Notably, the responses to load in the femoral and tibial cartilage revealed opposite effects. No significant differences were found in response to load between the two visits. This is the first study that analyzed the effects of acute loading on T1ρ and T2* measurements in human femoral and tibial cartilage separately. The results suggest the effect of acute compressive loading on T1ρ and T2* was: 1) opposite in the femoral and tibial cartilage; 2) larger in contacting regions than in non-contacting regions of the femoral cartilage; and 3) not different visit-to-visit., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)