Reliable drug concentration measurements at the target site are increasingly demanded and can be achieved by microdialysis. The aim of this pilot study was to demonstrate the proof of principle of long-term subcutaneous microdialysis in humans. For long-term microdialysis, a special setting implementing both concentric and linear catheters has been developed ensuring good clinical practice compliance, tolerability, and convenience for participants and personnel. As a model compound, moderately lipophilic voriconazole was selected as a well-characterized drug in in vitro microdialysis experiments. Multiple in vivo relative recovery (RR) determinations for microdialysis were performed by retrodialysis during the entire study (n = 48 samples). Continuous microdialysis was successfully applied and well tolerated over 87 h in three adults for the first time. RR revealed low intra-individual (coefficient of variation (CV) = 4.4-12.5%) and inter-individual variability (CV = 4.3-12.5%) across all samples and catheters. Lower RR values were consistently determined for linear catheters. One catheter leakage was managed without an impact on the reliability of the RR values. Overall, RR values were calculated to be 73.3% (linear: CV = 18.5%, n = 23) and 84.9% (concentric: CV = 5.6%, n = 23). Long-term microdialysis application over almost 4 days was feasible by reliable multiple RR (proof of principle), well tolerated, and reduced the burden in humans avoiding several catheter insertions, thereby allowing to monitor concentration-time courses continuously. Moreover, a moderately lipophilic drug has been proven suitable for in vivo microdialysis, as previously suggested by in vitro microdialysis.