Stratum corneum (SC) hydration is partially regulated by water-soluble molecules, natural moisturizing factor (NMF) that is associated with the corneocytes. Routine water exposure, e.g., bathing, may deplete NMF and alter the SC water-handling properties. We determined the effects of bathing and solvent extraction on the volar forearm skin of eleven healthy volunteers. Acetone/ether (A/E) was used to remove surface and upper SC lipids. Adjacent sites were soaked for ten minutes or treated with the A/E-plus-soak combination. Subsequently, an NMF formulation was applied to the treated sites, and transepidermal water loss (TEWL), hydration, and moisture accumulation rate (MAT) were measured. A/E extraction increased TEWL, but did not effect MAT. Soaking produced a short-term increase in TEWL, followed by a decrease, and substantially reduced MAT, an effect that was maintained for five hours. NMF application significantly decreased TEWL and significantly increased MAT for all sites. The replacement experiment suggests that the MAT reduction occurred as a result of extraction of hygroscopic NMF components. The effects of soaking and NMF application are more readily detected by the MAT technique, whereas TEWL is more sensitive to A/E extraction. The results support the use of multiple assessments of barrier function and raise questions about the effects of cumulative repeated water exposure on SC function.