Background: Eating frequency (EF) has been associated with generalized obesity. Aim: We aimed to prospectively investigate potential associations of frequency of eating episodes with regional fat layers. Design: EF was evaluated at baseline in 115 subjects free of clinically overt cardiovascular disease (54 6 9.1 years, 70 women) in a prospective, observational study. Methods: Metabolic parameters known to be associated with dietary factors and anthropometric markers including ultrasound assessment of subcutaneous (Smin) and pre-peritoneal (Pmax) fat and their ratio Smin/Pmax (AFI) were evaluated at baseline and at follow-up, 5 years later. Results: EF at baseline positively correlated with Pmax, even after adjustment for potential confounders. EF above median was also an independent predictor for Pmax (beta coefficient=-0.192, P = 0.037) and AFI (beta coefficient = 0.199, P = 0.049) at follow up. Multivariable linear mixed models analysis demonstrated that subjects with increased EF presented a lower progression rate of Pmax (beta=-0.452, P = 0.006) and a higher progression rate of AFI (beta = 0.563, P = 0.003) over time, independently of age, sex, progression of BMI, energy intake, smoking and changes in parameters of glucose metabolism. Conclusions: High EF is associated with lower progression rate of pre-peritoneal fat accumulation. Future interventional studies should further investigate the clinical utility of these findings.