Background Microdialysis-based glucose sensors have recently been introduced for monitoring glucose levels in diabetic patients. The flow rate by which the fluid sample is pumped through the microdialysis catheter varies in different studies. Aim To study the effects of various flow rates on glucose and its metabolites sampled by microdialysis during an oral glucose tolerance test. Material, methods Glucose, lactate, pyruvate and glycerol were measured with microdialysis in interstitial fluid of subcutaneous adipose tissue in twelve healthy young subjects before and during an oral glucose tolerance test using four different flow rates (0.3, 1, 2 and 5 μL/min) and a 30 mm dialysis membrane. Results At the basal fasting state the dialysate glucose obtained by 0.3 μL/min was equal to capillary glucose concentration. A decrease in dialysate glucose levels during the basal state was observed for higher flow rates but not for 0.3 μL/min, which indicates a depleting effect. The relative increase after OGTT was similar for capillary glucose and flow rate 0.3 μL/min but not for higher flow rates. Conclusion The low microdialysis flow rate (0.3 μL/min) facilitates the capture of true interstitial glucose concentrations during glucose fluctuations. Thus this low flow rate is preferred in studies of local tissue metabolism.