The response to loading of human articular cartilage as assessed by magnetic resonance imaging (MRI) remains to be defined in relation to histology and biomechanics. Therefore, an MRI-compatible whole-knee joint loading device for the functional in situ assessment of cartilage was developed and validated in this study. A formalin-fixed human knee was scanned by computed tomography in its native configuration and digitally processed to create femoral and tibial bone models. The bone models were covered by artificial femoral and tibial articular cartilage layers in their native configuration using cartilage-mimicking polyvinyl siloxane. A standardized defect of 8 mm diameter was created within the artificial cartilage layer at the central medial femoral condyle, into which native cartilage samples of similar dimensions were placed. After describing its design and specifications, the comprehensive validation of the device was performed using a hydraulic force gauge and digital electronic pressure-sensitive sensors. Displacement-controlled quasi-static uniaxial loading to 2.5 mm and 5.0 mm of the mobile tibia versus the immobile femur resulted in forces of N and N (on the entire joint) and local pressures of MPa and MPa (at the site of the cartilage sample). Upon confirming the MRI compatibility of the set-up, the response to loading of macroscopically intact human articular cartilage samples () was assessed on a clinical 3.0-T MR imaging system using clinical standard proton-density turbo-spin echo sequences and T2-weighted multi-spin echo sequences. Serial imaging was performed at the unloaded state and at consecutive loading positions (i.e. at and . Biomechanical unconfined compression testing (Young’s modulus) and histological assessment (Mankin score) served as the standards of reference. All samples were histologically intact (Mankin score, ) and biomechanically reasonably homogeneous (Young’s modulus, MPa). They could be visualized in their entirety by MRI and significant decreases in sample height [: mm; : mm; : mm; (repeated-measures ANOVA)] as well as pronounced T2 signal decay indicative of tissue pressurization were found as a function of compressive loading. In conclusion, our compression device has been validated for the noninvasive response-to-loading assessment of human articular cartilage by MRI in a close-to-physiological experimental setting. Thus, in a basic research context cartilage may be functionally evaluated beyond mere static analysis and in reference to histology and biomechanics.