Microdialysis is a sampling method that permits measurement of hormones, drugs, and other lower molecular weight compounds present in interstitial fluid. We developed a straightforward mathematical model that predicts a linear relationship between the reciprocal dialysate concentration of the analyte from the interstitium and perfusion rate, permitting estimation of the interstitial concentration by extrapolation to zero perfusion rate. Conversely, linearity between the reciprocal dialysate concentration of internal standard added to the perfusion medium (retrodialysis), and the reciprocal perfusion rate, is predicted. In nine healthy volunteers, interstitial norepinephrine (NE) was estimated by NE measurements in microdialysates obtained from skeletal muscle and adipose subcutaneous tissue, using sodium salicylate (Sal) in the perfusion buffer as internal standard, at perfusion rates of 2 and 5 mul/min. Comparison with microdialysis in vitro by immersing the probe in a large volume of buffer containing NE showed that the in vivo (retro)recovery of NE and Sal is almost exclusively determined by transport of NE through the interstitial space toward and Sal from the membrane and that membrane permeability itself plays a negligible role. This was supported by the observation that applying lower body negative pressure, a measure that is unlikely to affect membrane permeability, resulted in a significant (p < 0.05) decrease of Sal retrorecovery from muscle interstitium. This validated new model significantly adds insight into the factors determining recovery of substances from the interstitium in microdialysis and provides a simpler alternative to previous approaches for estimation of interstitial concentrations.