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The aim of this study was to differentiate septic from non-septic arthritis by measuring lactate concentration with 1H magnetic resonance spectroscopy (HMRS) and by estimating total protein content with the assessment of T2 values.In 30 patients with acute arthritis, synovial fluid was aspirated. Lactate concentrations were analyzed with single voxel HMRS at 1.5 T. T2 relaxation times were mapped with a multi-spin echo sequence. All samples underwent microbiological testing and routine laboratory analysis to quantify lactate concentration and total protein content. Values obtained in septic and non-septic arthritis were compared with a Mann-Whitney U test.Synovial fluid from patients with septic arthritis (n = 10) had higher concentrations of lactate (11.4 ± 4.0 mmol/L) and higher total protein content (51.8 ± 10.7 g/L) than fluid obtained in non-septic arthritis (n = 20; 5.2 ± 1.1 mmol/L and 40.4 ± 6.9 g/L, respectively, p < 0.001 and <0.01, respectively). Measured lactate concentrations and T2 relaxation times (as an indicator of total protein content) were moderately correlated to laboratory-confirmed lactate concentration (r2 = 0.71) and total protein content (r2 = 0.73). Markedly increased lactate concentrations (>6 mmol/L) in combination with low T2 values (<550 ms) identify septic arthritis with a sensitivity of 70% and a specificity of 89%.Spectroscopic measurements of lactate concentration in combination with the estimation of protein content using T2 may be of value in the differentiation of septic from non-septic arthritis.