Burnett CM, Grobe JL. Direct calorimetry identifies deficiencies in respirometry for the determination of resting metabolic rate in C57Bl/6 and FVB mice. Am J Physiol Endocrinol Metab 305: E916 -E924, 2013. First published August 20, 2013; doi:10.1152/ajpendo.00387.2013.-Substantial research efforts have been aimed at identifying novel targets to increase resting metabolic rate (RMR) as an adjunct approach to the treatment of obesity. Respirometry (one form of "indirect calorimetry") is unquestionably the dominant technique used in the obesity research field to assess RMR in vivo, although this method relies upon a lengthy list of assumptions that are likely to be violated in pharmacologically or genetically manipulated animals. A "total" calorimeter, including a gradient layer direct calorimeter coupled to a conventional respirometer, was used to test the accuracy of respirometric-based estimations of RMR in laboratory mice (Mus musculus Linnaeus) of the C57Bl/6 and FVB background strains. Using this combined calorimeter, we determined that respirometry underestimates RMR of untreated 9-to 12-wk-old male mice by ϳ10 -12%. Quantitative and qualitative differences resulted between methods for untreated C57Bl/6 and FVB mice, C57Bl/6 mice treated with ketamine-xylazine anesthesia, and FVB mice with genetic deletion of the angiotensin II type 2 receptor. We conclude that respirometric methods underestimate RMR in mice in a magnitude that is similar to or greater than the desired RMR effects of novel therapeutics. Sole reliance upon respirometry to assess RMR in mice may lead to false quantitative and qualitative conclusions regarding the effects of novel interventions. Increased use of direct calorimetry for the assessment of RMR and confirmation of respirometry results and the reexamination of previously discarded potential obesity therapeutics are warranted. metabolic rate; thermogenesis; heat; calorimetry; respirometry CLINICAL TREATMENT OF OBESITY is currently restricted to behavioral modification (e.g., exercise) and a short list of pharmaceutical agents (Orlistat, Qsymia, and Belviq). These three FDA-approved compounds/mixtures all function by reducing caloric intake/uptake. Interestingly, no safe or efficacious pharmaceutical treatments have been developed to increase resting metabolic rate (RMR), which could be used as an adjunct or alternative therapy to the intake-suppressing drugs currently available. In the 1930s, a compound that relieves the mitochondrial proton gradient called 2,4-dinitrophenol (DNP) was used extensively as a pharmacological stimulator of metabolic rate. Despite the great effectiveness of this compound to increase resting metabolism, use of DNP can result in cataracts and fatal hyperthermia, and therefore, it is no longer considered safe for human use (2). Substantial ongoing work in the field is aimed at understanding the mechanisms of RMR regulation, but useful pharmacological targets remain elusive. We hypothesize that a major problem facing the field is an inadequate ability to acc...