Metacarpophalangeal (MCP) joint proximal bone-cartilage specimens from the fourth digit were collected from repetitively flexed and non-flexed (control) paws of four New Zealand White rabbits. The specimens were cryo-fractured to reveal a sagittal cut containing the cartilage zones of different collagen microstructure. Nanoindentation, Fourier Transform infrared microspectroscopic imaging (FTIRMI), and histology were performed on a region of interest (ROI) ∼400 microns wide and through the thickness of the cartilage with two goals in mind: (1) to examine the effect of collagen network structure (random in the mid zone versus organized in the deep zone) on the biomechanical and biochemical properties of cartilage; and (2) to understand the changes in these properties due to physical forces. We found that zone microstructure significantly affected the measurement of the local relaxed modulus measured by nanoindentation. The deep zone had a higher modulus than the mid zone (Wilcoxon paired test, p<0.05). We also found that flexion significantly decreased the proteoglycan content in both the mid and deep zones (Wilcoxon paired test, p<0.05), suggesting indirect repetitive loading in the rabbit paw can be damaging to the joint via down regulating proteoglycan synthesis in the mid and deep zone cartilage. This is the first study to simultaneously report the local zone-specific mechanical and biochemical properties in the rabbit joint flexion model.