Fig. 2: Effect of housing temperature on energy expenditure, respiratory exchange ratio, food intake, water intake, and activity level in adult normal-weight male mice and DIO male mice.

Male (C57BL/6J, 20 weeks) DIO mice were housed individually in metabolic cages at 22 °C for a week preceding study initiation. Mice had ad libitum access to 45% HFD. After acclimatization, baseline data was collected for two days. Hereafter, temperature was increased in 2 °C increments every other day at 0600 h (start of light phase). Data are presented as means ± SEM and dark phase (1800-0600h) is indicated with grey boxes. a Energy expenditure (kcal/h), b total energy expenditures at different temperatures (kcal/24 h), c respiratory exchange ratios (VCO₂/VO₂: scale 0.7–1.0), d average RER (VCO₂/VO₂) during light and dark phase (zero-value defined as 0.7). e Cumulative food intakes (g), f total food intakes during 24 h, g cumulative water intake (mL), h total water intakes during 24 h, i cumulative activity level (m), and j total activity levels (m/24 h). Mice were at the indicated temperatures for 48 h. Data shown for 24, 26, 28, and 30 °C are from the last 24 h of each period. Mice remained on 45% HFD until end of study. Statistical significance was tested by One-way ANOVA for repeated measurements followed by Tukey multiple comparison test. Stars indicate significance from initial 22 °C values, hatches indicate significance between other groups as indicated. *P < 0.05, ***P < 0.001, ****P < 0.0001. Averages were calculated over the entire experimental period (0–192 h). n = 7.