Adequate sleep is essential for the biological maintenance of physical energy. Lack of sleep can affect thinking, lead to emotional anxiety, reduce immunity, and interfere with endocrine and metabolic processes, leading to disease. Previous studies have focused on long‐term sleep deprivation and the risk of cancer, heart disease, diabetes, and obesity. However, systematic metabolomics analyses of blood, heart, liver, spleen, kidney, brown adipose tissue, and fecal granules have not been performed. This study aims to systematically assess the metabolic changes in the target organs caused by sleep deprivation in vivo, to search for differential metabolites and the involved metabolic pathways, to further understand the impact of sleep deprivation on health, and to provide strong evidence for the need for early intervention.