Estimation of resting metabolic rate (RMR) is an important step for prescribing an individual’s energy intake. The purpose of this study was to evaluate the validity of portable indirect calorimeters and RMR prediction equations in muscular physique athletes. Twenty-seven males (n = 17; body mass index (BMI): 28.8 ± 2.0 kg/m²; body fat: 12.5% ± 2.7%) and females (n = 10; BMI: 22.8 ± 1.6 kg/m²; body fat: 19.2% ± 3.4%) were evaluated. The reference RMR value was obtained from the ParvoMedics TrueOne 2400 indirect calorimeter, and the Cosmed Fitmate and Breezing Metabolism Tracker provided additional RMR estimates. Existing RMR prediction equations based on body weight (BW) or dual-energy X-ray absorptiometry fat-free mass (FFM) were also evaluated. Errors in RMR estimates were assessed using validity statistics, including t tests with Bonferroni correction, linear regression, and calculation of the standard error of the estimate, total error, and 95% limits of agreement. Additionally, new prediction equations based on BW (RMR (kcal/day) = 24.8 × BW (kg) + 10) and FFM (RMR (kcal/day) = 25.9 × FFM (kg) + 284) were developed using stepwise linear regression and evaluated using leave-one-out cross-validation. Nearly all existing BW- and FFM-based prediction equations, as well as the Breezing Tracker, did not exhibit acceptable validity and typically underestimated RMR. The ten Haaf and Weijs (PLoS ONE, 9: e1084602014 (2014)) and Cunningham (1980) (Am. J. Clin. Nutr. 33: 2372–2374 (1980)) FFM-based equations may produce acceptable RMR estimates, although the Cosmed Fitmate and newly developed BW- and FFM-based equations may be most suitable for RMR estimation in male and female physique athletes. Future research should provide additional external cross-validation of the newly developed equations to refine the ability to predict RMR in physique athletes.