Human bile samples are commonly used in biliary research; however, the optimal sampling technique is not known. The current study examines whether bile obtained prior to operative manipulation of the gallbladder differs in composition from samples obtained after cholecystectomy. Gallbladder bile samples were obtained from 26 patients undergoing cholecystectomy. An initial sample was obtained prior to manipulation or devascularization of the gallbladder, and a second sample was obtained after the removal of the gallbladder from the operative field. Gallbladder bile pH and total protein were significantly increased in the postcholecystectomy samples. Bile obtained after cholecystectomy also contained significantly less phospholipid. Gallbladder bile cholesterol, total bile acids, bilirubin, ionized and total calcium, cholesterol saturation index, and total lipids were similar between techniques. These results indicate that manipulation of the gallbladder during cholecystectomy produces alterations in gallbladder bile composition. These results also emphasize the need for consistent sampling technique when obtaining samples for biliary research.