Plasma leucine concentration increases with short-term fasting in normal humans. In previous studies using an 18-h constant infusion of [2H3]leucine, a 15% decrease in the rate of appearance (Ra) of leucine was observed between 15 and 30 h of fasting. However, incorporation of labeled leucine into and subsequent release from body protein could result in an apparent decrease in leucine Ra. The present studies were undertaken to determine the rate of leucine N and carbon flux in short-term fasted human subjects in using an experimental design that would minimize potential recycling of label. Six normal subjects were infused with [15N]- and [2H3]leucine for two 4-h periods (0600 to 1000 h and 2000 to 2400 h) during a 30-h fast. Between the 15th and 30th h of fasting, plasma leucine concentration (102 +/- 10 to 169 +/- 18 microM, P less than 0.01) and leucine C (1.46 +/- 0.05 to 1.64 +/- 0.10 mumol X kg-1 X min-1, P less than 0.05) and leucine N (1.93 +/- 0.14 to 2.49 +/- 0.26 mumol X kg-1 X min-1, P less than 0.05) flux increased, whereas the metabolic clearance rates of leucine C and N decreased (P less than 0.01 and less than 0.05, respectively). Therefore, when isotope recycling is minimized with short periods of isotope infusion, leucine flux increases between 15 and 30 h of fasting. Because the only source of leucine in the postabsorptive periods is body protein, we conclude that the rate of whole-body proteolysis is increased in short-term fasting in humans.(ABSTRACT TRUNCATED AT 250 WORDS)