The most common approach for estimating substrate rate of appearance (R_a) is use of the single-pool model first proposed by R. W. Steele, J. S. Wall, R. C. DeBodo, and N. Altszuler. (Am. J. Physiol. 187: 15–24, 1956). To overcome the model error during highly non-steady-state conditions due to the assumption of a constant volume of distribution (V), two strategies have been proposed: 1) use of a variable tracer infusion rate to minimize tracer-to-tracee ratio (TTR) variations (fixed-volume approach) or2) use of two tracers of the same substrate with one infused at a constant rate and the other at a variable rate (variable-volume approach or approach of T. Issekutz, R. Issekutz, and D. Elahi. Can. J. Physiol. Pharmacol. 52: 215–224, 1974). The goal of this study was to compare the results of these two strategies for the analysis of the kinetics of glycerol and glucose under the non-steady-state condition created by a constant infusion of epinephrine (50 ng ⋅ kg⁻¹ ⋅ min⁻¹) with the traditional approach of Steele et al., which uses a constant infusion and fixed volume. The results showed that for glucose and glycerol the estimates of R_aobtained with the constant and the variable tracer infusion rate and the equation of Steele et al. were comparable. The variable tracer infusion approach was less sensitive to the choice of V in estimating R_a for glycerol and glucose, although the advantage of changing the tracer infusion rate was greater for glucose than for glycerol. The model of Issekutz et al. showed instability when the ratio TTR₁/TTR₂approaches a constant value, and the model is more sensitive to measurement error than the constant-volume model for glucose and glycerol. We conclude that the one-tracer constant-infusion technique is sufficient in most cases for glycerol, whereas the one-tracer variable-infusion technique is preferable for glucose. Reasonable values for glucose R_a can be obtained with the constant-infusion technique if V = 145 ml/kg.