The aim of this study was to use whole body calorimetry to directly measure the change in body heat content (DeltaH(b)) during steady-state exercise and compare these values with those estimated using thermometry. The thermometry models tested were the traditional two-compartment model of "core" and "shell" temperatures, and a three-compartment model of "core," "muscle," and "shell" temperatures; with individual compartments within each model weighted for their relative influence upon DeltaH(b) by coefficients subject to a nonnegative and a sum-to-one constraint. Fifty-two participants performed 90 min of moderate-intensity exercise (40% of Vo(2 peak)) on a cycle ergometer in the Snellen air calorimeter, at regulated air temperatures of 24 degrees C or 30 degrees C and a relative humidity of either 30% or 60%. The "core" compartment was represented by temperatures measured in the esophagus (T(es)), rectum (T(re)), and aural canal (T(au)), while the "muscle" compartment was represented by regional muscle temperature measured in the vastus lateralis (T(vl)), triceps brachii (T(tb)), and upper trapezius (T(ut)). The "shell" compartment was represented by the weighted mean of 12 skin temperatures (T(sk)). The whole body calorimetry data were used to derive optimally fitting two- and three-compartment thermometry models. The traditional two-compartment model was found to be statistically biased, systematically underestimating DeltaH(b) by 15.5% (SD 31.3) at 24 degrees C and by 35.5% (SD 21.9) at 30 degrees C. The three-compartment model showed no such bias, yielding a more precise estimate of DeltaH(b) as evidenced by a mean estimation error of 1.1% (SD 29.5) at 24 degrees C and 5.4% (SD 30.0) at 30 degrees C with an adjusted R(2) of 0.48 and 0.51, respectively. It is concluded that a major source of error in the estimation of DeltaH(b) using the traditional two-compartment thermometry model is the lack of an expression independently representing the heat storage in muscle during exercise.