Background: The ‘new normal’ during COVID-19 outbreak has presented unique dermatologic challenges related to respiratory protective equipment (RPE)-related skin conditions. Objectives: The study aimed to evaluate the effects of RPE on the skin barrier by measuring various physiological properties of the skin. Methods: 20 healthy healthcare workers were enrolled. Skin parameters including skin hydration, transepidermal water loss (TEWL), erythema, sebum secretion, pH, and skin temperature were measured in the RPE-covered and RPE-uncovered areas of the face 4 and 8 hours after wearing RPE and 14 hours after not wearing RPE. Results: Skin hydration, TEWL, erythema, pH, and skin temperature significantly increased over time on the RPE-covered skin. By contrast, in the RPE-uncovered areas, skin hydration decreased and TEWL, erythema, and pH showed minimal changes over time. Based on the repeated-measure analysis, the changes in skin physiological properties over time were significantly different between RPE-covered and RPE-uncovered areas. Conclusion: We observed that skin physiological characteristics change with the prolonged use of RPE. These changes may lead to various adverse skin reactions after long-term use.