Collagen and glycosaminoglycans (GAGs) constituting the extracellular matrix (ECM) may limit the space available and thus exclude macromolecules from a fraction of the interstitial fluid phase. This exclusion phenomenon is of importance for transcapillary fluid and solute exchange. We examined the range of interstitial exclusion in skin by using probes within a span of molecular weights and electrical charge, and also to tested if a change in interstitial composition occurring as a consequence of aging affected exclusion. To this end we used a novel approach, involving the exact determination of albumin concentration and mass in interstitial fluid and tissue eluate by high performance liquid chromatography and thereafter expressing the corresponding numbers relative to albumin for a set of probe proteins assessed by quantitative proteomics. There was a highly significant positive correlation between probe Stokes-Einstein radius and fractional excluded volume, and oppositely, a statistically significant, negative correlation between probe isoelectric point and exclusion for proteins with comparable size. Aging resulted in a significant reduction in skin hydration and sulphated GAGs, and a corresponding reduced fractional excluded volume for albumin and the other macromolecular probes. We have furthermore tested whether a high salt diet results in changes in biophysical properties of the ECM including lymph drainage from the interstitium. Overall, our findings suggest that the changes in the ECM in aged skin may result in delayed adjustments of fluid perturbations and reduced ability for salt storage.