Background:In contrast to the physiological expectation, observational studies show that greater protein intake is associated with subsequent body weight (BW) gain. An increase in fat-free mass (FFM) due to anabolic effects of protein could explain this.Objective:To examine associations between protein intake and subsequent changes in fat mass (FM) and FFM in longitudinal, observational data.Design:A health examination, including measures of FM and FFM by bioelectrical impedance at baseline and follow-up six years later, was conducted. Diet history interviews (DHI) were performed, and 24-hour urinary nitrogen collection at baseline was done. In total, 330 participants with DHI, of whom 227 had validated and complete 24-hour urine collection, were analyzed. Macronutrient energy substitution models were used.Results:Mean estimated protein intake was 14.6 E% from DHI and 11.3 E% from urinary nitrogen. Estimated from DHI, FM increased 46 gram/year with every 1 E% protein substituted for fat (95%CI: 13, 79; P=0.006) and FFM increased 15 gram/year (1, 30; P=0.046). Results were similar in other substitution models. Estimated from urinary nitrogen, FM increased 53 gram/year with 1 E% protein substituted for other macronutrients (24, 81; p<0.0005), and FFM increased 18 gram/year (6, 31; P=0.004).Conclusion:Within a habitual range, a greater protein intake was associated with BW gain, mostly in FM. This is in contrast to the expectations based on physiological and clinical trials, and calls for a better understanding of how habitual dietary protein influences long-term energy balance, versus how greater changes in dietary proteins may influence short-term energy balance.International Journal of Obesity accepted article preview online, 20 May 2014; doi:10.1038/ijo.2014.80.