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Mechanical stress is an important external factor effecting the development and maintenance of articular cartilage. The metabolite profile of diseased cartilage has been well studied but there is limited information about the variation in metabolite profile of healthy cartilage. With the importance of load in maintaining healthy cartilage, regional differences in metabolite profile associated with differences in load may provide information on how load contributes to the maintenance of healthy cartilage. HR-MAS NMR spectroscopy allows the assessment of tissue samples without modification and was used for assessing the difference in metabolic profile between the load bearing and non-load bearing regions of the bovine articular cartilage. In this preliminary study, we examined cartilage from tibia and femur of four knee joints. Sixteen pairs of 1D-NOESY spectra were acquired. Principle component analysis (PCA) identified chemical shifts responsible for variance. SBASE (AMIX) and the Human Metabolome Database were used in conjunction with previous reported cartilage data for identifying metabolites associated with the PCA results. The major contributors to load-related differences in metabolite profile were N-acetyl groups, lactate and phosphocholine peaks. Integrals of these regions were further analysed using a Student's t-test. In load bearing cartilage regions. N-acetyl groups and phosphocholine were found at significantly higher concentration (p < 0.05 and p < 0.005, respectively) in both femur and tibia, while lactate was reduced in load bearing cartilage (p < 0.005). The results of this pilot HR-MAS NMR study demonstrate its ability to provide useful metabolite information for healthy cartilage.