OBJECTIVE To help standardize methodology for intravenous glucose tolerance testing in preclinical IDDM by comparing a 30-s bolus and a 3-min infusion of glucose. RESEARCH DESIGN AND METHODS We tested 20 healthy nondiabetic adults at four centers (in Seattle, Boston, Melbourne, and London). Each subject had four intravenous glucose tolerance tests (two bolus and two infusion). The acute insulin response to glucose was calculated as the mean of the 1' + 3', the mean of 1' to 10', or as the integrated area from 0 to 10'. Glucose and insulin profiles and intrasubject coefficient of variation were compared. RESULTS With the infusion protocol, the 1' insulin was significantly higher, resulting in a higher acute insulin response to glucose when calculated as 1' + 3' (525 +/- 66 vs. 376 +/- 35 pM, P < 0.004). When calculated over 10 min, however, the acute insulin response to glucose was not different between protocols. In addition, the intrasubject coefficient of variation was significantly better when calculated over 10 min in both protocols, but no significant differences were noted between the bolus and infusion (infusion: AIRg [area from 0 to 10'] 10.4 +/- 2.1% vs. AIRg [1' + 3'] 14.9 +/- 2.8%, P < 0.007; bolus: AIRg [area from 0 to 10'] 14.6 +/- 2.8% vs. AIRg [1' + 3'] 19.8 +/- 3.5%, P < 0.007). Comparison of the insulin assays between the four centers showed close correlation and gave indistinguishable results in terms of within-subject coefficient of variation. Glucose profiles were similar in both protocols. Although the glucose values were lower with the bolus protocol from 4' to 40', the rate of fall from 10 to 30' (and thus the rate of glucose disposal) was indistinguishable between the two. CONCLUSIONS These data suggest that neither protocol gives significant advantage over the other. However, to allow comparison of the acute insulin response to glucose between different protocols used in centers around the world, the ICARUS 3-min infusion protocol is recommended, with acute insulin response to glucose calculated over 10 min after the end of glucose administration; this reduces the within-subject coefficient of variation and provides similar acute insulin response to glucose with both protocols.