Scratching and skin barrier dysfunctions are pivotal features and therapeutic targets of atopic dermatitis (AD); however, time-of-day-dependent variations of these characteristics remain unclear. NC/Tnd mice have been shown to exhibit severe scratching behavior and skin barrier disruption together with the development of spontaneous atopic dermatitis when they are raised under air-uncontrolled environment. In the present study, time-of-day-dependent variations of scratching behavior and transepidermal water loss (TEWL) were evaluated in NC/Tnd mice that developed moderate to severe AD. Analysis of the mice for 24 hr revealed that scratching frequency and duration were increased from in the afternoon to the nocturnal period when locomotor activity was low, and scratching behavior was decreased in the morning. The highest scratching frequency and duration were 3.8- and 4.1-fold increases in the lowest scratching frequency and duration, respectively. In addition, TEWL on the dorsal skin lesion was decreased in the diurnal period, while that was increased in the nocturnal period. The highest TEWL was a 1.3-fold increase in the lowest TEWL. Significant daily variations were detected in scratching frequency and duration and TEWL. These results indicate that NC/Tnd mice are an appropriate mouse model to investigate time-of-day-dependent variations of scratching behavior and skin barrier dysfunctions associated with AD.