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Glucose is essential for numerous cellular functions due to its involvement in energy supply from early development to adulthood. In the lactating cow, glucose demands by the mammary gland significantly increase to support milk production when compared with other tissues. Thus, insufficient blood glucose levels might predispose dairy cows to metabolic disturbances. The intravenous glucose tolerance test (ivGTT) is a suitable tool to characterize glucose metabolism and insulin responses, but results must be reliable and repeatable. One factor influencing ivGTT is food deprivation period, which has been considered as an obligatory requirement before conduction of glucose tolerance studies in monogastric species, whereas it has been permissive in ruminants. The objective of this study was to determine the influence of 5 fasting periods (0, 12, 24, 36, and 48 h) on ivGTT traits and insulin responses in German Holstein heifers. A total of 140 tests were conducted in 28 females aged 12 to 19 mo. Blood samples were collected every 7 min within 1 h. Assessed glucose and insulin parameters included basal serum glucose and insulin concentration, maximum glucose and insulin concentration obtained between min 7 to 21, and concentrations at min 63 (last sampling) relative to glucose administration, glucose area equivalent (GA), glucose area under the curve (GAUC), insulin area equivalent (InsA), insulin area under the curve (InsAUC), and blood glucose half-life time (GHLT). Serum glucose and insulin concentration were measured according to the hexokinase colorimetric method and solid phase radio immunoassay, respectively. The generalized linear mixed model was used to test for significant differences in all glucose traits and insulin responses at different fasting periods. The model used season and weight as confounding variables. Glucose and insulin concentrations at 0, 7 to 21 (maximum concentration), and 63 min were affected by the duration of food deprivation. The GA, InsA, GAUC, InsAUC, and especially GHLT were also affected by fasting period. A positive linear relationship between GHLT and length of food deprivation was found. Significantly higher GA, GAUC, GHLT values, and glucose and insulin concentration at min 63 were obtained at increasing fasting periods. High intraclass correlation coefficient (0.48) was found for GHLT. The results demonstrate that GHLT might be used to reliably characterize an individual's glucose metabolism and the importance of standardizing food deprivation schedules when performing an ivGTT in nongestating Holstein heifers.