Five subjects underwent twenty electrically evoked maximal isometric contractions of the anterior tibialis muscle of both legs (n = 10), with limb blood flow occluded. Measurements of muscle high-energy phosphates (ATP, ADP and phosphocreatine (PCr)), lactate and pH were made using both 31P magnetic resonance spectroscopy (MRS) and the biochemical analysis of biopsy samples obtained from directly below the MRS surface coil. The resting PCr concentration determined using MRS was similar to that measured in the biopsy material. Following contraction, MRS showed a greater decrease in ATP concentration compared with biochemical analysis (P < 0.05), but the decrease in PCr was similar. Good agreement was found when comparing resting muscle pH estimated by the two methods. Post-exercise muscle pH was, however, consistently lower with MRS and consequently the accumulation of muscle lactate estimated using MRS was markedly greater than the corresponding biochemical measurement (P < 0.05). As a result, MRS revealed an approximately 30% greater anaerobic ATP turnover during contraction, although this just failed to reach statistical significance (P > 0.05). The results of the present study indicate that there is little difference in the muscle concentration of PCr estimated by the two methods, but that there are differences in the estimates of ATP, pH and lactate changes during contraction. This latter discrepancy may lead to greater estimates of ATP turnover being made as a result of MRS analysis.