Microdialysis presents the unique possibility to measure metabolite concentrations in human interstitial fluid. During exercise, the recovery of these metabolites should be precisely monitored since it is known to increase greatly with muscle blood flow. The loss of ethanol, perfused at low concentration, can be accurately measured and reflects the changes in dialysis conditions. We evaluated whether using the relationship determined in resting metabolic conditions between the loss of ethanol, as reference substance, and the recovery for lactate or glucose would allow us to calculate precisely the concentration of these substances and their variations during exercise. Using the new catheter calibration method (slope method), the error of estimation of lactate and glucose in vitro was limited to -0.6 (5.8)% and -0.7 (6.2)%, respectively. In resting human muscle, the slope method proved to be as accurate as an established calibration technique ("no net flux method") to evaluate interstitial lactate concentration [1.82 (0.58) vs 1.83 (0.47) mM, respectively]. During dynamic knee-extension exercise or light neuromuscular electrical stimulation, the estimated interstitial lactate and glucose concentrations varied differently, but their time course changes remained consistent with their respective plasma values. We conclude that, after an initial calibration step, the slope method allows accurate measurement of interstitial muscle metabolites and it could be used to monitor rapid metabolic changes during exercise.