Background: Minimally invasive glucose monitoring is an important tool for a better management of diabetes in order to avoid long-term complications. Methods: A vacuum operated ultrafiltration sampling unit for interstitial fluid (ISF) with integrated electroosmotic flow enhancement has been developed. The novel device was tested with three non-diabetic, healthy volunteers. Results: Flow measurements showed an increased ISF transport depending on the applied electrical current. Preliminary online glucose measurements showed reasonable results. Conclusion: Electroosmotically enhanced ISF harvesting seems to be a promising approach for generating minimally invasive glucose monitoring devices, although results have shown significant ISF flow for the applied microsensors.