Sleep loss induces performance impairment and fatigue. The reactivation of human herpesvirus-6, which is related to the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), is one candidate for use as an objective biomarker of fatigue. Phosphorylated eIF2α is a key regulator in integrated stress response (ISR), an intracellular stress response system. However, the relation between sleep/sleep loss and ISR is unclear. The purpose of the current study was to evaluate the effect of prolonged sleep deprivation and recovery sleep on ISR-related gene expression in rat liver. Eight-week-old male Sprague–Dawley rats were subjected to a 96-hour sleep deprivation using a flowerpot technique. The rats were sacrificed, and the liver was collected immediately or 6 or 72 h after the end of the sleep deprivation. RT-qPCR was used to analyze the expression levels of ISR-related gene transcripts in the rat liver. The transcript levels of the Atf3, Ddit3, Hmox-1, and Ppp15a1r genes were markedly increased early in the recovery sleep period after the termination of sleep deprivation. These results indicate that both activation and inactivation of ISRs in the rat liver occur simultaneously in the early phase of recovery sleep.