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Simple Summary The demands of modern society have made shift work a necessity. Night work is associated with an increased risk of metabolic problems such as obesity and diabetes, which is mainly due to the misalignment of circadian rhythms that play a crucial role in many biological processes. This study performed clinical, anthropometric, and molecular analyses on 40 hospital workers who work day or night. We demonstrated that night workers had increased glucose levels, triglycerides, waist circumference, and blood pressure compared to day workers. Surprisingly, we report that night workers have significant changes in the expression of circadian clock genes and an up-regulation of genes related to endoplasmic reticulum stress (ERS). These findings provide new insights into the effects of night shift work on the expression of circadian cycle genes and ERS activation, leading to metabolic stress and the development of metabolic diseases associated with night work. Abstract Night work has become necessary in our modern society. However, sleep deprivation induces a circadian misalignment that effectively contributes to the development of diseases associated with metabolic syndrome, such as obesity and diabetes. Here, we evaluated the pattern of circadian clock genes and endoplasmic reticulum stress (ERS) genes in addition to metabolic and anthropometric measures in subjects that work during a nocturnal period compared with day workers. We study 20 night workers (NW) and 20 day workers (DW) submitted to a work schedule of 12 h of work for 36 h of rest for at least 5 years in a hospital. The present report shows that NW have increased fasting blood glucose, glycated hemoglobin (HbA1c), triglycerides, and low-density lipoprotein (LDL)-cholesterol levels, and lower high-density lipoprotein (HDL)-cholesterol levels compared to DW. In addition, we observed that waist circumference (WC), waist–hip ratio (WHR), and systemic blood pressure are also increased in NW. Interestingly, gene expression analysis showed changes in CLOCK gene expression in peripheral blood mononuclear cells (PBMC) samples of NW compared to the DW, evidencing a peripheral circadian misalignment. This metabolic adaptation was accompanied by the up-regulation of many genes of ERS in NW. These findings support the hypothesis that night shift work results in disturbed glycemic and lipid control and affects the circadian cycle through the deregulation of peripheral CLOCK genes, which is possibly due to the activation of ERS. Thus, night work induces important metabolic changes that increase the risk of developing metabolic syndrome.