In-vivo assessments of intestinal permeability can be expensive and time consuming. Additionally, the correct choice of test molecules to use and the optimum sampling time under fasting situations needs be optimized. Fifteen unweaned Angus-Holstein bull calves (44.1 ± 2.0 kg and 14.7 ± 0.63 d) were randomly assigned to 1 of 3 treatments: Control (CT; n = 5): no fasting; fasted during 9 h (FAS9; n = 5); and fasted during 19 h (FAS19; n = 5). All calves were fed 2.5 L of MR and treatments were applied on d -1. Chromium-EDTA (Cr-EDTA), lactulose and D-mannitol were administered orally before blood collection. Samples were taken on d -4 and -1 before fasting and on d 0 and 2, at 60, 120, 180, and 240 minutes. To choose the best test molecule, correlations between serum concentration of the test molecules were used. To decide the optimum sampling time, data from d 0 were used to calculate area under the curve, then data were analyzed with mixed models with fasting degree and sampling time as fixed effects. Correlation between Cr-EDTA and D-mannitol was r= 0.92 (R2=84%), and correlation between Cr-EDTA and lactulose was r= 0.86 (R2=75%). Differences in AUC of Cr-EDTA between CT and t fasting treatments were observed up to 120 min. Differences in AUC of Cr-EDTA between FAS9 and FAS19 were observed later at 240 min. To optimize the intestinal permeability test, the use of only one test molecule might be sufficient. The Cr-EDTA was proposed to optimize the methodology due to price and simplicity of the analysis. The optimum sampling time after Cr-EDTA administration was 120 min when comparing fasting and control, and 240 min when comparing different fasting degrees.