Objective: To examine in humans the effects on serum lipids, lipoproteins and fat-soluble antioxidants of a daily consumption of 2.5 g plant stanols, consumed either once per day at lunch or divided over the three meals. Design: A randomized, double-blind, placebo-controlled, cross-over design. Subjects: Thirty-nine healthy normocholesterolemic or mildly hypercholesterolemic subjects participated. Interventions: Each subject consumed in random order; no plant stanols; 2.5 g plant stanols at lunch; and 2.5 g plant stanols divided over the three meals (0.42g at breakfast, 0.84g at lunch and 1.25 g at dinner, which is proportional to dietary cholesterol intake). Each period lasted 4 weeks. Plant stanols were esterified with fatty acids from low erucic rapeseed oil (LEAR) and incorporated into margarines or shortenings. Results: Consumption of 2.5g plant stanols at lunch results in a similar low-density lipoprotein (LDL)-cholesterol-lowering efficacy compared to consumption of 2.5 g plant stanols divided over the three meals (-0.29 mmol/l compared with the control period (P <0.001; 95% CI, -0.19 to -0.39 mmol/l) for the once per day diet and -0.31 mmol/l (P <0.001; 95% CI, -0.20 to -0.41mmol/l) for the three times per day period). High-density Lipoprotein (HDL) cholesterol and triacylglycerol concentrations did not change. After standardization for LDL cholesterol, the sum of the most lipophylic hydrocarbon carotenoids (ie alpha-carotene, beta-carotene and lycopene) in particular was slightly, though not significantly, lowered by -0.017 +/- 0.018 mu mol/mmol LDL cholesterol (P = 0.307) after the once per day period and by -0.032 +/- 0.016 mu mol/mmol LDL cholesterol (P = 0.049) after the three times per day period. Conclusions: Our findings suggest that for lowering LDL cholesterol concentrations it is not necessary to consume products rich in plant stanol ester at each meal or simultaneously with dietary cholesterol.