This study evaluated a zero-heat-flow (ZHF), non-invasive temperature probe for in- vivo measurement of resting muscle temperature for up to 2 cm below the skin surface. The ZHF probe works by preventing heat loss from the tissue below the probe by actively heating the tissue until no temperature gradient exists across the probe. The skin temperature under the probe is then used as an indicator of the muscle temperature below. Eight subjects sat for 130 min during exposure to 28 degrees C air. Vastus lateralis (lateral thigh) muscle temperature was measured non-invasively using a ZHF probe which covered an invasive multicouple probe (which measured tissue temperature 0.5 cm, 1 cm, 1.5 cm, and 2 cm below the skin) located 15 cm superior to the patella (T (covered)). T (covered) was evaluated against an uncovered control multicouple probe located 20 cm superior to the patella (T (uncovered)). Rectal temperature and lateral thigh skin temperature were also measured. Mean T (uncovered) (based on average temperatures at the 0.5 cm, 1 cm, 1.5 cm, and 2 cm depths) and Mean T (covered) were similar from time 0 min to 60 min. However, when the ZHF was turned on at 70 min, Mean T (covered) increased by 2.11 +/- 0.20 degrees C by 130 min, while T (uncovered) remained stable. The ZHF probe temperature was similar to T (covered) at 1 cm and after time 85 min, significantly higher than T (covered) at the 0.5 cm, 1.5 cm, and 2 cm depths; however from a physiological standpoint, the temperatures between the different depths and the ZHF probe could be considered uniform (< or =0.2 degrees C separation). Rectal and thigh skin temperatures were stable at 36.99 +/- 0.08 degrees C and 32.82 +/- 0.23 degrees C, respectively. In conclusion, the non-invasive ZHF probe temperature was similar to the T (covered) temperatures directly measured up to 2 cm beneath the surface of the thigh, but all T (covered) temperatures were not representative of the true muscle temperature up to 2 cm below the skin because the ZHF probe heated the muscle by 2.11 +/- 0.20 degrees C during its operation.