Technical Abstract: Little is known about how lactating women accommodate for their increased glucose demands during fasting to avoid maternal hypoglycemia. The objective of this study was to determine whether lactating women conserve plasma glucose by reducing maternal glucose utilization by increasing utilization of FFA, and ketone bodies and/or increasing gluconeogenesis and mammary gland hexoneogenesis. Six healthy exclusively breastfeeding women and six non-lactating controls were studied during 42 h of fasting and 6 h of re-feeding. Glucose and protein kinetic parameters were measured using stable isotopes, GCMS, and energy expenditure and substrate oxidation using indirect calorimetry. After 42 h of fasting, milk production decreased by 16% but remained within normal range. Glucose, insulin, and C-peptide concentrations decreased with the duration of fasting in both groups but were lower (P < 0.05) in lactating women. Glucagon, FFA, and beta-hydroxybutyrate concentrations increased with fasting time (P < 0.001) and were higher (P < 0.0001) in lactating women during both fasting and re-feeding. During 42 h of fasting, gluconeogenesis was higher in lactating women compared with non-lactating controls (7.7 +/- 0.4 vs. 6.5 +/- 0.2 umol.kg–1.min–1, P < 0.05), whereas glycogenolysis was suppressed to similar values (0.4 +/- 0.1 vs. 0.9 +/- 0.2 umol.kg–1.min–1, respectively). Mammary hexoneogenesis did not increase with the duration of fasting. Carbohydrate oxidation was lower and fat and protein oxidations higher (P < 0.05) in lactating women. In summary, lactating women are at risk for hypoglycemia if fasting is extended beyond 30 h. The extra glucose demands of extended fasting during lactation appear to be compensated by increasing gluconeogenesis associated with ketosis, decreasing carbohydrate oxidation, and increasing protein and FFA oxidations.