Background: Many laboratories offer glycemic index (GI) services. Objective: We assessed the performance of the method used to measure GI. Design: The GI of cheese-puffs and fruit-leather (centrally provided) was measured in 28 laboratories (n = 311 subjects) by using the FAO/WHO method. The laboratories reported the results of their calculations and sent the raw data for recalculation centrally. Results: Values for the incremental area under the curve (AUC) reported by 54% of the laboratories differed from central calculations. Because of this and other differences in data analysis, 19% of reported food GI values differed by > 5 units from those calculated centrally. GI values in individual subjects were unrelated to age, sex, ethnicity, body mass index, or AUC but were negatively related to within-individual variation (P = 0.033) expressed as the CV of the AUC for repeated reference food tests (refCV). The between-laboratory GI values (mean +/- SD) for cheese-puffs and fruit-leather were 74.3 +/- 10.5 and 33.2 +/- 7.2, respectively. The mean laboratory GI was related to refCV (P = 0.003) and the type of restrictions on alcohol consumption before the test (P = 0.006, r(2) = 0.509 for model). The within-laboratory SD of GI was related to refCV (P