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BACKGROUND: The flux of absorbed cholesterol is a controlling element in the regulation of cholesterol biosynthesis and catabolism. A review of 5 published methods to measure cholesterol absorption is presented, including 2 dual stable isotope approaches. The continuous dual isotope feeding procedure is accurate, but only suitable for small-scale studies. The blood-based dual stable isotope technique is the least invasive and complex procedure, but leads to large variations in individual (90%) and mean population values (24%-70%) for healthy subjects. The results may be partly determined by the experimental and analytical procedures.SOURCES OF MATERIAL: Fifteen blood-based dual stable isotope studies published between 1993 and 2013 have been analyzed. The results were related to the methodologies applied and evidence was sought for accordance to the test principles.FINDINGS: Seven different isotopic tracers, 3 cholesterol subcompartments in blood, and 6 mass spectrometry techniques were applied. The oral and intravenous test formulations were presented in only 1 study. Time points for blood sampling and methodologies for blood sample preparation and analysis were highly variable. No definite proofs were supplied for the fates of the oral and intravenous cholesterol tracers. Isotope enrichment measurements in free and total cholesterol in plasma and erythrocytes were never compared. Fractional cholesterol absorption rate values depend strongly on the mass spectrometry methodology. Dual-inlet isotope ratio mass spectrometry appears to be the method of choice.CONCLUSIONS: Dual stable isotope approaches require validation and standardization of administration and analysis procedures. A control group must always be included to correct for methodological differences. (C) 2015 National Lipid Association. All rights reserved.