Short high-voltage pulses have recently been shown to dramatically increase and expedite transdermal drug transport via a mechanism hypothesized to involve electroporation. This study addresses tolerance issues of the method in vivo in hairless rat. Chromametry, transepidermal water loss (TEWL), laser Doppler flowmetry (LDF) and corneometry were jointly used for noninvasive sensing of skin biophysical parameters. Slight increases in skin redness, TEWL and LDF values followed the application of electric pulses. The changes in skin capacitance were nonsignificant. The magnitude of the alterations depended on the electrical features of the pulses. When compared to iontophoresis, high-voltage pulses did not induce stronger alterations of skin functions. This report provides the first in vivo demonstration of the safety of the high-voltage pulses proposed for transdermal delivery.