Lack of sleep is a problem in today's society, and many people are concerned about changes in their outward appearance due to lack of sleep. People generally come up with some noticeable skin attributes as symptoms of sleep deficiency including rough, dull, and dry skin as well as droopy eyelids and dark eye circles. Several previous reports also suggested that poor sleep could affect the skin condition. The purpose of this study was to evaluate the effect of one night of sleep deprivation on various skin biophysical properties. Twenty four healthy females participated in a study of one night of sleep deprivation. Subjects were kept awake for one night in a laboratory with controlled temperature and relative humidity. The skin condition of each subject was evaluated after normal sleep pattern and after one night of sleep deprivation. The measured skin biophysical parameters included transepidermal water loss, facial pore size, and skin tone, hydration, elasticity, desquamation, translucency, and blood flow. The cheek, eye, and lip areas were evaluated. After one night of sleep deprivation, multiple skin biophysical parameters showed changes when compared to the baseline measurements. A significant decrease in skin hydration and impaired barrier function were observed (p < 0.05). Decreased hydration led to decreased skin elasticity and translucency and increased skin scaling (p < 0.05). Facial pores were more conspicuous, and skin lightness decreased significantly (p < 0.05). Furthermore, skin blood flow decreased prominently (p < 0.05). The results demonstrated that the skin features that are recognized as symptoms of sleep deficiency actually showed remarkable differences after a period of sleep deprivation, and some of these features were confirmed in the eye, lip, as well as the cheek areas. This study revealed a significant association between sleep deprivation and skin biophysical properties by scientific measuring.