BACKGROUND: The protein leverage hypothesis requires specific evidence that protein intake is regulated more strongly than energy intake. OBJECTIVE: The objective was to determine ad libitum energy intake, body weight changes, and appetite profile in response to protein-to-carbohydrate + fat ratio over 12 consecutive days and in relation to age, sex, BMI, and type of protein. DESIGN: A 12-d randomized crossover study was performed in 40 men and 39 women [mean +/- SD age: 34.0 +/- 17.6 y; BMI (in kg/m(2)): 23.7 +/- 3.4] with the use of diets containing 5%, 15%, and 30% of energy from protein from a milk or plant source. RESULTS: Protein-content effects did not differ by age, sex, BMI, or type of protein. Total energy intake was significantly lower in the high-protein (7.21 +/- 3.08 MJ/d) condition than in the low-protein (9.33 +/- 3.52 MJ/d) and normal-protein (9.62 +/- 3.51 MJ/d) conditions (P = 0.001), which was predominantly the result of a lower energy intake from meals (P = 0.001). Protein intake varied directly according to the amount of protein in the diet (P = 0.001). The AUC of visual analog scale appetite ratings did not differ significantly, yet fluctuations in hunger (P = 0.019) and desire to eat (P = 0.026) over the day were attenuated in the high-protein condition compared with the normal-protein condition. CONCLUSIONS: We found evidence to support the protein leverage hypothesis in that individuals underate relative to energy balance from diets containing a higher protein-to-carbohydrate + fat ratio. No evidence for protein leverage effects from diets containing a lower ratio of protein to carbohydrate + fat was obtained. It remains to be shown whether a relatively low protein intake would cause overeating or would be the effect of overeating of carbohydrate and fat. The study was registered at clinicaltrials.gov as NCT01320189.