The relationship between physical activity levels (PAL) and the presence of cardiovascular disease (CVD) risk factors such as anthropometric and biochemical indices and heat shock proteins 27 antibody (anti-HSP-27) concentration, and serum inflammatory markers, was investigated in the MASHAD cohort study.The overall study population consisted of 9,684 subjects (3,858 men, 5,826 women) with a mean age of 47.73 ± 8.08 to 48.87 ± 9.26 years respectively. They were divided into four categories based on their PAL. Biochemical parameters were determined for all participants. Also, serum anti-HSP-27 levels were measured using an in-house enzyme-linked immune sorbent assay method. Multiple regression analysis was used to explore the association between the anti-HSP antibody titers and physical activity after adjusting for confounding factors. The level of statistical significance was set at p  0.05).We found that PAL was significantly associated with several established CVD risk factors. Also, the level of anti-HSP-27 was lower in individuals with moderate and high PAL.