The mechanical properties of the Stratum Corneum (SC) have been studied by different authors at the macroscopic level, but the modification of its ultra structure during mechanical extension remains unknown. Moreover, little is described about the effect of the mechanical stress on SC barrier function. In this study, we have examined the SC structure changes, at the molecular level, during uniaxial tensile experiments. This was performed on isolated SC samples using Raman spectroscopy. We could identify the strain status of the analyzed samples by using combination of Raman spectra and Partial Least Squares processing. In addition, this approach provided information about lipids and proteins behavior during the sample extension. The structure of the intercellular lipids bilayer became less organized up to ~9% deformation. For higher strains, a plateau corresponding to the minimum organization is observed till the complete failure of the sample. In the same time, protein structures including desmosomes, were characterized by monotonic secondary structure modifications for deformations up to ~9% followed by a plateau. These observations are relevantly demonstrating the effect of extension on the skin barrier state. Such an approach could be objectively used for clinical applications to evaluate skin discomfort degree and skin elastic behavior. This could therefore help with proof of efficacy for cosmetic and dermatologic products. Copyright © 2013 John Wiley & Sons, Ltd.