Fasting gluconeogenesis (GNG) 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 (fr)GNG can be calculated using the ratio of C5 to C2 enrichment or the ratio of C5 to 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 18 h, we studied the effects of 3-, 5-, and 15-h 2H2O incubation periods on 1) the equilibration of plasma 2H2O and C2 glucose enrichment, 2) the measurement of frGNG, and 3) C5 labeling of hepatic glycogen after 1 mg of glucagon administration. After 3-h 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 15-h 2H2O incubation, plasma 2H2O and C2 were equilibrated, and frGNG C5/2H2O and C5/C2 were similar, as was GNG calculated with C5/2H2O and C5/C2. After glucagon administration, no difference of C5 enrichment was found between 3, 5, and 15 h of 2H2O incubation. In conclusion, for reliable measurement of GNG in healthy subjects with C5/2H2O incubation periods longer than 3 h are required. After 5- and 15-h 2H2O incubation, GNG can be reliably measured with C5/2H2O. Gluconeogenetic labeling of glycogen did not affect the results after 3, 5, or 15 h of 2H2O incubation.