The study was an open, prospective, randomized cross-over design to determine if dehydration during fasting increases lipid concentrations. Fifteen healthy subjects participated, 1 of whom did not complete the study. The subjects fasted once with no fluid replacement and once with salt and water supplementation. Following both fasts, blood was drawn for lipid assessments. Compared to fasting with fluid and salt replacement, fasting with no fluids was associated with higher (mean, 95% confidence interval) total serum cholesterol (8.1%, 4.3-11.9%), HDL cholesterol (7.5%, 1.8-13.1%), LDL cholesterol (10.5%, 2.2-18.8%), apolipoprotein A-1 (8.9%, 5.0-12.8%), and apolipoprotein B (10.5%, 5.2-15.8%). The change in serum triglycerides was not statistically significant (12.4%,-0.5-25.3%). There was a greater reduction in body weight during fasting with fluid restriction compared to fasting with salt and water supplementation (1.8%, 1.3-2.2%). Fasting with fluid restriction results in significantly higher lipid levels and, therefore, variation in hydration of patients could contribute to fluctuation in lipid levels of patients. Care should be taken to ensure that patients are in a standard state of hydration during assessment of lipid levels. We recommend: 1) that patients fast no longer than 12 h, and 2) that, during fasting, patients avoid unnecessary physical activity, avoid hot dry environments, ensure a liberal intake of water, and avoid diuretic substances such as caffeine.