University of Melbourne Department of Medicine (Austin Health/Northern Health), Heidelberg Repatriation Hospital, Heidelberg Heights, Victoria, Australia Submitted 21 July 2008 ; accepted in final form 18 September 2008 The objective of this study was to determine the optimal conditions under which to assess glucose tolerance in chow- and high-fat-fed C57BL/6J mice. Mice were fed either chow or high-fat diet for 8 wk. Variables tested were fasting duration (0-, 3-, 6-, and 24-h and overnight fasting), route of administration (intraperitoneal vs. oral) load of glucose given (2, 1, or 0.5 g/kg and fixed 50-mg dose), and state of consciousness. Basal glucose concentrations were increased in high-fat- compared with chow-fed mice following 6 h of fasting (9.1 ± 0.3 vs. 7.9 ± 0.4 mmol/l P = 0.01). Glucose tolerance was most different and therefore significant ( P = 0.001) in high-fat-fed mice after 6 h of fasting (1,973 ± 96 vs. 1,248 ± 83 mmol·l –1 ·120 min –1 ). The difference in glucose tolerance was greater following an OGTT (142%), in contrast to an IPGTT, with a 127% difference between high fat and chow. We also found that administering 2 g/kg of glucose resulted in a greater level of significance ( P = 0.0008) in glucose intolerance in high-fat- compared with chow-fed mice. A fixed dose of 50 mg glucose regardless of body weight was enough to show glucose intolerance in high-fat- vs. chow-fed mice. Finally, high-fat-fed mice showed glucose intolerance compared with their chow-fed counterparts whether they were tested under conscious or anesthetized conditions. We conclude that 2 g/kg glucose administered orally following 6 h of fasting is best to assess glucose tolerance in mice under these conditions. intraperitoneal glucose tolerance test; oral glucose tolerance test; fasting; high-fat feeding; C57BL/6J Address for reprint requests and other correspondence: S. Andrikopoulos, Univ. of Melbourne, Dept. of Medicine (AH/NH), Heidelberg Repatriation Hospital, 300 Waterdale Road, Heidelberg Heights, Victoria, Australia (e-mail: sof{at}unimelb.edu.au )