The effects of diet composition on obesity and related life-style diseases currently receive considerable attention. Protein-rich diets seem to increase fat loss and reduce the weight regain that often follows weight loss. These effects have mostly been attributed to the satiating efficiency of protein and the thermogenic effect of a protein-rich diet. However, the effects of dietary protein content on basal and maximal metabolic rates have not been examined in detail. Adult female mice (C57BL/6JBom) were fed one of the isocaloric diets with either high (5:3), standard (1:2) or low (1:7) protein to carbohydrate ratio ad libitum for 34–47 days. Thereafter, metabolic rates were determined during fasting, using open flow respirometry. Basal metabolic rate (BMR) was recorded within the thermoneutral zone, and maximum metabolic rate was induced by cold exposure. BMR of mice on the protein-rich diet was almost 11% higher than BMR in the other groups (VO₂:29.2±0.8 and 26.4±0.8mL·min^-1·kg^-1, respectively). While diet did not significantly affect maximum metabolic rate of the mice, the average spontaneous energy expenditure during overnight measurements increased with increasing protein content of the diets (VO₂: 37.9±1.1, 39.8±1.1 and 42.5±0.2mL· min^-1·kg^-1, respectively). Our study shows that elevated basal metabolic rate and increased average energy expenditure are likely to contribute to the increased mass loss in response to elevated protein levels in the diet during dietary interventions.