The procedure of indirect calorimetry is often combined with the hyperinsulinemic, euglycemic clamp technique so that intracellular rates of glucose oxidation (Gox), fat oxidation (Fox), and energy expenditure (EE) can be determined at different insulin concentrations and rates of whole-body glucose uptake. In order to perform these calculations, rates of protein oxidation (Pox) must be known and are usually estimated from urinary nitrogen (N) excretion. The use of urinary N assumes that this measurement accurately reflects Pox and is unaltered by the glucose clamp technique. To examine these assumptions and determine potential errors in rates of Gox, Fox, and EE with this method, eight healthy subjects each had basal urinary N excretion determined on 4 different days and during a 300 pmol/m2/min hyperinsulinemic, euglycemic clamp. Mean basal urinary N excretion was 6.4 +/- 1.6 mg/min. Within individuals, basal urinary N was highly variable on the 4 different days with a mean coefficient of variation (CV) of 36% +/- 18%. Over the range of basal respiratory quotient (RQ) values in this study (0.78 to 0.85) the day-to-day variation in basal urinary N resulted in potential errors of 11% to 23% for Gox, 16% to 24% for Fox, but minimal effects (less than or equal to 1%) on EE. During the hyperinsulinemic, euglycemic clamp, RQ increased to 0.95 or greater, while urinary N excretion, rather than decreasing as expected, increased by 47% (6.4 +/- 1.6 to 9.4 +/- 2.8 mg/min) due in part to increases in urea clearance from 37.5 +/- 6.7 to 75.2 +/- 12.4 mL/min (P less than .025). This increased urinary N excretion had minimal influence on Fox and EE, but underestimated Gox by up to 5% at RQ less than 0.95. A more accurate estimate of urinary N excretion during hyperinsulinemic clamps may be obtained by correcting for changes in urea clearance. These results indicate that basal urinary N excretion is highly variable and influenced by hyperinsulinemic glucose clamps. Thus, urinary N excretion, particularly during the basal state, may not accurately reflect changes in Pox and can lead to substantial errors in Gox and Fox.