Background:Working outside daylight hours (7 am to 7 pm) is called shift work. Shift work is a common practice in many industries and factories such as steel industries, petroleum industries, power plants, and in some services such as medicine and nursing and police forces, in which professionals provide services during day and night.Objectives:Considering the contradictory reports of different studies, we decided to evaluate the effect of shift work on cholesterol and triglyceride (TG) levels through a historical cohort on steel industry workers.Patients and Methods:This retrospective cohort study was performed on all the staff of Isfahan’s Mobarakeh Steel Company between years 2002 and 2011. There were 5773 participants in this study. Data were collected from the medical records of the staff using the census method. For analysis of data, generalized estimating equation (GEE) regression was used.Results:The results showed a significant difference in cholesterol levels between shift workers and day workers on the first observation (P < 0.001), yet no such difference was observed for TG (P = 0.853). Moreover, the results showed that the variables of age, work experience and BMI were not similar between shift workers and day workers. Therefore, to remove the effect of such variables, we used GEE regression. Despite the borderline difference of cholesterol between regular shift workers and day workers, this correlation was not statistically significant (P = 0.051). The results for TG also showed no correlation with shift work.Conclusions:According to the findings of this study, there is no relationship between shift work and changes in serum TG and cholesterol. The lack of relationship can be due to shift plans for shift workers, nutrition, or the “Healthy Heart project” at Isfahan Mobarakeh Steel Company.