The purpose of the present study was to use the microdialysis technique to determine skeletal muscle interstitial glucose and lactate concentrations during dynamic incremental exercise in humans. Microdialysis probes were inserted into the vastus lateralis muscle, and subjects performed knee extensor exercise at workloads of 10, 20, 30, 40, and 50 W. The in vivo probe recoveries determined at rest by the internal reference method for glucose and lactate were 28.7 ± 2.5 and 32.0 ± 2.7%, respectively. As exercise intensity increased, probe recovery also increased, and at the highest workload probe recovery for glucose (61.0 ± 3.9%) and lactate (66.3 ± 3.6%) had more than doubled. At rest the interstitial glucose concentration (3.5 ± 0.2 mM) was lower than both the arterial (5.6 ± 0.2 mM) and venous (5.3 ± 0.3 mM) plasma water glucose levels. The interstitial glucose levels remained lower (P < 0.05) than the arterial and venous plasma water glucose concentrations during exercise at all intensities and at 10, 20, 30, and 50 W, respectively. At rest the interstitial lactate concentration (2.5 ± 0.2 mM) was higher (P < 0.05) than both the arterial (0.9 ± 0.2 mM) and venous (1.1 ± 0.2 mM) plasma water lactate levels. This relationship was maintained (P < 0.05) during exercise at workloads of 10, 20, and 30 W. These data suggest that interstitial glucose delivery at rest is flow limited and that during exercise changes in the interstitial concentrations of glucose and lactate mirror the changes observed in the venous plasma water compartments. Furthermore, skeletal muscle contraction results in an increase in the diffusion coefficient of glucose and lactate within the interstitial space as reflected by an elevation in probe recovery during exercise.