The main function of articular cartilage is to distribute loads and provide low friction for the opposing surfaces in synovial joints. Biphasic lubrication provided by high fluid load support due to relative motion of the contact surfaces has been widely accepted as the main lubrication mode in diarthrodial joints. However, assessment of chondrocyte response to mechanical loads typically employed nonphysiological uniaxial loads with static contact area. This study aimed to introduce a more physiologically relevant loading protocol for in vitro mechanobiological testing of cartilage explants. Finite element analysis was conducted to examine the biomechanical response of cartilage to two different loading regimes, biaxial loading, that permits migrating contact area, and unconfined uniaxial cyclic compression, traditionally used in mechanobiological experiments. Results predicted in this study showed that continuous tissue rehydration provided by relative surface motion maintained constant fluid pressure and tissue strains through the simulation. On the contrary, due to rapid tissue consolidation predicted in cyclic compression simulation, fluid pressure and transverse strain were reduced by 19% and 26%, respectively. Furthermore, relative surface motion simulation resulted in depth-dependent distribution of fluid pressure and tissue strains while unconfined uniaxial cyclic compression produced nearly uniform fluid pressure through the depth but higher at the center of the sample. Based on the results obtained from this study and since sliding contact occurs in vivo, this physiological loading mode should be considered in assessing biomechanical and mechanobiological cartilage behavior.