It is not yet clear to what extent the physiologic regulatory mechanisms that maintain core body temperature are reflected by changes in resting energy expenditure (REE). Particularly, in indirect calorimetry with a canopy, the effects of short-term temperature exposures have not yet been investigated. This can be of relevance for the determination of REE in practice.This randomized crossover study investigated the influence of a 30-min exposure to 18 °C (cool room temperature), 22 °C (room temperature), 28 °C (thermoneutral zone), and 38 °C (heat) on REE determined by assessing the inhaled oxygen volume and the exhaled carbon dioxide volume via indirect calorimetry on 4 consecutive days.In total, 32 metabolically healthy participants [16 males and 16 females, age: 25 ± 3 y, body mass index (BMI): 22.4 ± 1.6 kg/m2] were included in the study after screening examination.Lean body mass, ambient temperature, and heart rate were the most important determinants (all P < 0.001) and explained 61.3% of the variance in REE. A multivariate linear mixed model analysis revealed that lean mass (15.87 ± 3.66; P < 0.001) and ambient temperature (P = 0.001) significantly influenced REE. REE significantly differed between 18 and 28 °C (18 °C: +96 ± 24 kcal/24 h; P < 0.001), 22 and 28 °C (22 °C: +73 ± 24 kcal/24 h; P = 0.003), and 18 and 38 °C (18 °C: +57 ± 23 kcal/24 h; P = 0.016).Effects of ambient temperature on REE, especially cold, are detectable after only brief exposure, emphasizing the importance of performing indirect calorimetry with a canopy under controlled environmental conditions. The study was registered at clinicaltrials.gov as NCT05505240 (Influence of Ambient Temperature on Resting Energy Expenditure of Healthy Adults - Full Text View - ClinicalTrials.gov).