Fasting gluconeogenesis is often quantified using the ²H₂O technique which is based on plasma ²H₂O enrichment and ensuing enrichment of plasma glucose at the C5 and C2 positions. Fractional gluconeogenesis (frGNG) can be calculated using the ratio of C5 and C2 enrichment or the ratio of C5 and plasma ²H₂O enrichment. For the latter equilibration of ²H₂O and C2 is required. The optimal equilibration period of ²H₂O and C2 remains to be elucidated. In six healthy male subjects, fasted for 18h, we studied the effects of 3, 5 and 15h ²H₂O incubation periods on 1) the equilibration of plasma ²H₂O and C2 glucose enrichment, 2) the measurement of fractional gluconeogenesis and 3) C5 labeling of hepatic glycogen after 1 mg glucagon administration . After 3h ²H₂O incubation, plasma ²H₂O and C2 were not equilibrated, frGNG C5/²H₂O and C5/C2 were also different as was gluconeogenesis calculated with C5/²H₂O and C5/C2. After 5 and 15h ²H₂O incubation, plasma ²H₂O and C2 were equilibrated, frGNG C5/²H₂O and C5/C2 were similar, as was gluconeogenesis calculated with C5/²H₂O and C5/C2. After glucagon administration no difference of C5 enrichment was found between 3, 5 and 15h ²H₂O incubation. In conclusion, for reliable measurement of gluconeogenesis in healthy subjects with C5/²H₂O incubation periods longer than 3h are required. After 5 and 15h of ²H₂O incubation, gluconeogenesis can be reliably measured with C5/²H₂O. Gluconeogenetic labeling of glycogen did not affect the results after 3, 5 or 15h ²H₂O incubation.