The purpose of this study was to assess a novel technique for quantifying in vivo muscle protein metabolism and phenylalanine transport in septic patients and normal volunteers and thereby assess the influence of sepsis on muscle protein kinetics. In patients resuscitated from sepsis, blood flow and edema may influence the extent of muscle loss. Six adult patients septic from pneumonia underwent a study protocol consisting of infusion of isotopic phenylalanine, indocyanine green dye, and sodium bromide; biopsies of skeletal muscle; and sampling from the femoral artery, vein, and interstitial fluid. Study results demonstrate a substantial net catabolism of muscle, an accelerated flux of phenylalanine, and an increased leg blood flow for septic patients compared with normal volunteers. For septic patients and normal volunteers, the rate of phenylalanine transport through the interstitium was rate limiting for the movement of phenylalanine between vasculature and muscle. Measurements demonstrate a concentration gradient of phenylalanine favoring the net efflux of amino acids from the leg in the septic patients. Despite whole body edema, the extracellular fluid volume within muscle of septic patients was similar to normal. These findings demonstrate that the extent of muscle loss in critically ill patients results from the net increase in the rate of muscle protein breakdown, which subsequently drives amino acids through the interstitial compartment down their concentration gradient. Therefore, any effective therapy to correct illness-induced muscle catabolism should be directed at altering the rates of breakdown and synthesis of muscle protein and are not likely related to tissue edema.