To compare the metabolic profiles of synovial fluid (SF) from patients with anterior cruciate ligament tears and hemarthrosis (HA) with that of normal controls, using 1H NMR spectroscopy (NMRS). Synovial fluid was collected from eleven patients undergoing arthroscopic debridement within fourteen days following an anterior cruciate ligament (ACL) tear and hemarthrosis. Ten additional SF samples were obtained from the knees of osteoarthritis-free volunteers to serve as normal controls. The relative concentrations of twenty-eight endogenous SF metabolites (hydroxybutyrate, acetate, acetoacetate, acetone, alanine, arginine, choline, citrate, creatine, creatinine, formate, glucose, glutamate, glutamine, glycerol, glycine, histidine, isoleucine, lactate, leucine, lysine, phenylalanine, proline, pyruvate, threonine, tyrosine, valine, and the mobile components of glycoproteins and lipids) were evaluated using NMRS and quantified using CHENOMX metabolomics analysis software. Mean differences between groups were evaluated with t-tests controlling for multiple comparisons at an overall error rate of 0.10. Statistically significant increases in the levels of glucose, choline, the branched-chain amino acids leucine, isoleucine, and valine, and the mobile components of N-acetyl glycoproteins and lipids were observed in ACL/HA SF as compared with normal controls; lactate levels were reduced. Marked changes occur in the metabolic profiles of human knee fluid following ACL injury and hemarthrosis, suggestive of increased demand and accompanying inflammatory response; potentially increased lipid and glucose metabolism; and possible hyaluronan degradation within the joint following trauma.