The application of short-duration high-voltage pulses to the skin has been shown to enhance transdermal drug delivery by several orders of magnitude and to transiently permeabilize cells in tissue. Both exponentially decaying (ED) pulses and square wave (SW) pulses have been applied. The latter have also been used for electrochemotherapy. To date, their effect on skin integrity has not been analyzed. The scope of this work was (i) to investigate the effect induced by SW pulses on the stratum corneum and the skin, (ii) to evaluate the safety issue associated with electroporation, (iii) to contribute to the understanding of drug transport. Biophysical techniques (transepidermal water loss, chromametry, impedance and laser Doppler velocimetry or imaging measurement) and histological methods were combined to provide a global picture of the effects. Ten SW pulses applied to the skin induced a mild impairment of the skin barrier function and a dramatic decrease in skin resistance. These changes were reversible. A transient decrease (<5 min) in blood flow was observed. Neither inflammation, nor necroses were observed. These studies confirm the tolerance of the skin to square wave pulses in vivo.