Gastrointestinal luminal contents may interfere with concentration analysis of non-absorbable dyes. However, non-absorbable markers are broadly used for studies of gastric emptying rates of nutrient solutions. This prompted us to evaluate the properties of non-absorbable markers to mark such nutrient solutions. In vitro concentrations of polyethylene glycol, phenol red, dextran blue, two anthroquinone dyes and inulin were determined spectrophotometrically in the presence or absence of a formula diet, single compounds of the diet or an oligo-peptide diet, and the reproducibility and validity of the analyses were evaluated. The presence of the formula diet or the oligopeptide diet seriously impaired the analyses of marker concentrations, whereas single nutrient compounds did not uniformly interfere. The analysis of polyethylene glycol and phenol red concentrations was impaired by proteins, while the analysis of inulin concentration was impaired by carbohydrates. Dextran blue and the anthroquinones were completely eliminated by protein-precipitation procedures. In conclusion, phenol red and polyethylene glycol should only be used as marker substances for protein-free meals or nutrient solutions, while inulin should not be used with meals or nutrient solutions containing carbohydrates. Marker dilution techniques cannot be recommended for measurements of gastric emptying rates of complete meals.