Night and shift work might be risk factors for metabolic and cardiovascular disorders due to interference with diet, circadian metabolic rhythms, and lifestyle. The relationship between permanent night work and metabolic and cardiovascular risk factors was explored in a retrospective longitudinal study of workers employed in a large municipal enterprise in charge of street cleaning and domestic waste collection. All subjects who had worked night shifts between 1976 and 2007 as hand sweepers, motor sweepers, and delivery tricar drivers were compared with subjects who always worked the same jobs but on day shifts. From the periodical medical surveillance files, we identified 488 male workers who had been examined on average five times (minimum 2, maximum 14) during the study period, for a total of 2,328 medical examinations; 157 always had worked day shifts, 12 always the night shift, and 319 both (initially day and subsequently night shifts). Their age ranged from 22 to 62 yrs, and work experience varied from 1 to 28 yrs. Lifestyle habits (smoking, alcohol consumption), body mass index, serum glucose, total cholesterol, tryglicerides, hepatic enzymes, blood pressure, resting electrocardiogram, diabetes, coronary heart disease, hypertension, and related drugs were taken into consideration for the analysis. We used generalized estimating equations (GEE) models (exchangeable correlation matrix) to analyze the relationship between night work and health effects while accounting for within-subject correlations and adjusting for study period, job, age, and lifestyle variables. As a whole, night workers smoked more and had significantly higher BMI, serum total cholesterol, and triglycerides than day workers. Both the inter-individual comparison between day and night workers and the intra-individual comparison among the workers, who were day workers at the beginning of their employment and later became night workers, showed a significant increase in BMI, total cholesterol, and tryglicerides associated with night work. No consistent effect was seen on fasting glucose, hepatic enzymes, and blood pressure, whereas a higher incidence of coronary heart disease was recorded in night workers.