OBJECTIVE There are conflicting data in the literature regarding the reproducibility of the gallbladder ejection fraction (GBEF) measured by fatty meal cholescintigraphy (CS). We aimed to test the reproducibility of GBEF measured by fatty meal CS. METHODS Thirty-five subjects (25 healthy volunteers and 10 patients with chronic abdominal pain) underwent fatty meal CS twice in order to measure GBEF1 and GBEF2. The healthy volunteers underwent a repeat scan within 1-13 months from the first scan. The patients underwent a repeat scan within 1-4 years from the first scan and were not found to have chronic acalculous cholecystitis (CAC). Our standard fatty meal was composed of a 60-g Snickers chocolate bar and 200 ml full-fat yogurt. RESULTS The mean ± SD values for GBEF1 and GBEF2 were 52 ± 17% and 52 ± 16%, respectively. There was a direct linear correlation between the values of GBEF1 and GBEF2 for the subjects, with a correlation coefficient of 0.509 (p = 0.002). Subgroup data analysis of the volunteer group showed that there was significant linear correlation between volunteer values of GBEF1 and GBEF2, with a correlation coefficient of 0.473 (p = 0.017). Subgroup data analysis of the non-CAC patient group showed no significant correlation between patient values of GBEF1 and GBEF2, likely due to limited sample size. CONCLUSIONS This study showed that fatty meal CS is a reliable test in gallbladder motility evaluation and that GBEF measured by fatty meal CS is reproducible.