We investigated the applicability of the bicarbonate stable isotope dilution technique for accurate determination of 24-h energy expenditure in humans and in reference to the conduct of short-term (less than or equal to 8 h) metabolic studies. Five healthy adult subjects consumed for 4 d a standard diet providing similar to 188 kJ.kg(-1).d(-1) and 1.0 g.kg(-1).d(-1) of egg protein. From d 4 at 1800 h to d 5 at 1800 h, a 24 h metabolic study, combining indirect calorimetry with an intravenous infusion of C-13-labeled sodium bicarbonate, was performed under standardized conditions of 12 h fasting-12 h feeding. ''Measured'' CO2 production (VCO2) (indirect calorimetry) over 24 h was not significantly different from ''predicted'' VCO2 (bicarbonate dilution) (218.31 +/- 20.91 vs. 221.51 +/- 19.44 mmol CO2.kg(-1).d(-1) respectively) (P = 0.34). Further, 24-h VCO2 and energy expenditure were determined by extrapolating from ''predicted'' VCO2 during the last hour of fasting (15th hour following last meal) and the fifth hour of the small hourly meal-feeding phase. The maximum difference for an individual subject between the above-calculated 24-h energy expenditure and that from 24-h indirect calorimetry measurements was <4%. These results support use of the present experimental protocol, and the bicarbonate dilution technique, lasting Is h, to obtain reliable quantitative estimates of 24-h CO2 production and energy expenditure in healthy adult humans.