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Allergic disorders such as atopic dermatitis (AD) are strongly associated with an impairment of the epithelial barrier, in which tight junctions and/or filaggrin expression can be defective. Skin barrier assessment shows potential to be clinically useful for prediction of disease development, improved and earlier diagnosis, lesion follow-up and therapy evaluation. This study aimed to establish a method to directly assess the in vivo status of epithelial barrier using electrical impedance spectroscopy (EIS).