Dead Sea mud (DSM) is commonly used by patients with various skin conditions because of its contents of healing elements. No study was published to show whether DSM application weakens or strengthens skin barrier function. In this study, we investigated the impact of 30-minute single application of various types of DSM ("As Is" mud, mud with extra Dead Sea salt, and over-the-shelf mud) on the barrier function of normal skin. The influence of 30-minute application of various types of DSM was investigated noninvasively on skin barrier properties of healthy female adult volunteers (n = 75) on predetermined circular areas. Skin hydration, transepidermal water loss (TEWL), erythema and melanin levels, and skin pH were measured directly, 30 minutes, and 60 minutes after mud removal. Thirty-minute single application of DSM was well tolerated with short-lived moisturizing effects, which was enhanced by the presence of humectant ingredients, and with no negative impact on barrier integrity, pH, and erythema and melanin levels.