To define the relationship between the respiratory quotient (RQ) and energy intake (EI) and to determine the impact of spontaneous locomotor activity (LMA) in the development of diet-induced obesity (DIO), we fed C57BL/6 mice a high-fat diet (HFD) for either 4 days or 17 wk and analyzed them using indirect calorimetry. Importantly, changes in body mass during calorimetry (DeltaM(b)) significantly covaried with RQ and EI; adjusting the data for DeltaM(b) permitted an analysis of the energy-balanced state. The 24-h RQ strongly predicted 24-h EI, and the slope of this relationship was diet dependent (HFD or chow) but independent of the HFD feeding period. Early-stage DIO was characterized by dark-period hyperphagia and fat storage, offset by greater light-period lipid oxidation; later stage DIO mice had a milder hyperphagia and lower substrate flexibility. Consequently, whereas 24-h RQ equaled the food quotient of the HFD in both early- and late-stage DIO, the range of RQ values was negatively correlated with, and mostly explained by, 24-h EI only in late-stage DIO. Lean and early-stage DIO mice had similar LMA values that were reduced in late-stage DIO. However, LMA significantly explained variance in total energy expenditure (EE) in only early-stage DIO mice. This indicated that the link between LMA and EE was a transient adaptive response to early DIO, whereas the later loss of LMA did not explain body weight gain in C57BL/6 DIO mice.