Resting metabolic rate (RMR) studies frequently involve genetically-manipulated mice and high fat diets (HFD). We hypothesize that the use of inadequate methods impedes the identification of novel regulators of RMR. This idea was tested by simultaneously measuring RMR by direct calorimetry and respirometry in C57BL/6J mice fed chow, 45% HFD, and then returned to chow. Comparing results during chow feeding uncovered an underestimation of RMR by respirometry (0.010 ± 0.001 kcal/h, P < 0.05), which is equivalent in magnitude to ∼2% of total daily caloric turnover. RMR during 45% HFD feeding was increased by respirometry (+0.013 ± 0.003 kcal/h, P < 0.05), but not direct calorimetry (+0.001 ± 0.002 kcal/h). Both methods indicated that return to chow reduced RMR compared to HFD, though direct calorimetry indicated a reduction below the initial chow fed state (−0.019 ± 0.004 kcal/h versus baseline, P < 0.05) that was not detected by respirometry (−0.003 ± 0.002 kcal/h versus baseline). These results highlight method-specific interpretations of the effects of dietary interventions upon RMR in mice, and prompt the reevaluation of preclinical screening methods used to identify novel RMR modulators.