The reliability and stability of plasma lipid biomarkers and their association with dietary fat intake were evaluated among 48 subjects who were randomly chosen from the participants of a validation study of the population-based cohort, the Shanghai Men's Health Study (SMHS). Four spot blood samples, one taken each season, were measured for total cholesterol, triglyceride, HDL-cholesterol, and LDL-cholesterol levels. The reliability and stability of these measurements were assessed by intraclass correlation coefficients (ICC) and by the correlations between a randomly chosen measurement with the mean of measurements across seasons using a bootstrap approach. The median levels for total cholesterol, triglycerides, HDL-cholesterol, and LDL-cholesterol were 177.5, 164.5, 41.0, and 102.5 (mg/dl), respectively. The ICCs of the biomarkers ranged from 0.58 (LDL-cholesterol) to 0.83 (HDL-cholesterol). The correlation between randomly chosen spot measurements and the mean measurement were 0.91, 0.86, 0.93, and 0.83 for total cholesterol, triglycerides, HDL-cholesterol, and LDL-cholesterol, respectively. The correlations of lipid biomarkers with dietary fat intake and other lifestyle factors were comparable to other previous reports. In conclusion, this study suggests that measurements of lipid biomarkers from a single spot blood sample are a good representation of the average blood levels of these biomarkers in the study population and could be a useful tool for epidemiological studies.