Background: Firm conclusions regarding the etiology of heat exposure responses among animals are difficult to draw due to different experimental designs and methodological confounders, such as environmental chamber set-up and heating rates. The purpose of this investigation was to 1) validate the heat test protocol for mice models via cage location and orientation; and to 2) determine the maximum number of cages that can be used without compromising individual heating rates. Methods: A mice temperature transponder (G2 E-Mitter, Mini Meter, Respironics) was centrally positioned inside each mice cage set in the environmental chamber (Thermo Scientific Forma, Model 3961). Two cage orientations (adjacent, left-to-right and parallel, front-to-back) with 3 set-ups (top shelf, bottom shelf and both shelves) using 2 and 4 cages were examined in triplicate and averaged. Transponders equilibrated at 21.5℃ for 5 min, then exposed to 39.5℃ for a minimum of 60 min. Results: A major finding was that adjacent (L-R) top shelf set-up had the smallest temperature difference throughout the heat test (Δ = 0.43℃ vs. Δ = 2.2℃) and at minute 60 (Δ = 0.2℃ vs. Δ = 1.8℃). Both orientations for the bottom shelf set-up had a slower rise in temperature (0.04℃·min-1) than other set-ups (0.3℃·min-1). Using both shelves, top shelf cages were consistently warmer than bottom shelf cages (1.0℃ - 3.6℃) for both orientations. Conclusions: We strongly suggest using an adjacent (L-R) top shelf set-up since it enabled uniform chamber heating rates and standardized heat exposure. Bottom shelf is not recommended for use due to poor heating rate performance. Since an increased number of cages may obstruct heat flow patterns, a one shelf set-up with 2 cages should be used.