A cross-sectional study was conducted to investigate dietary intake, behavioral habits, and clinical and metabolic differences in night workers compared to day workers and to evaluate the metabolic differences associated with diet and body habits that occur between these two groups. Dietary habits, biological parameters, and health status were collected in 1,200 night workers and in an equal number of day workers, matched for gender, age, and socioeconomic status. Our findings demonstrated that night workers had poorer dietary habits and metabolic profile compared to day workers with a similar overall health status. These differences were associated with a higher prevalence of some cardiovascular risk factors such as smoking and obesity.