The doubly labeled water method is a noninvasive technique for measurement of rates of CO2 production and total energy expenditure in free-living human subjects. The experimental protocol used in validation studies usually involves prolonged fasting before and after the isotope dose is given to start the study, although it is not clear whether this intrusive aspect of the method is necessary. We investigated this issue in four healthy adults [3 women and 1 man: age 29 +/- 2 (SD) yr, body mass index 22 +/- 2.7 kg.m2] with monitored constant physical activity who underwent two doubly labeled water studies that differed only in the duration of fasting before and after isotope dosing (either 6 h of fasting before and 5 h after dosing or 0.5 h before and 3 h afterward). No significant difference between the two measurements was found in the rate constants for isotope disappearance, the ratio of the dilution spaces of the isotopes, or CO2 production rate. These results indicate that prolonged fasting before and after isotope administration should not be necessary in doubly labeled water studies.