Objective: Energy restriction decreases fat mass and fat-free mass. Our aim was to prevent the latter using type and timing of protein nutrition as tools. Methods: Young male Wistar rats were given a high-energy diet for 5 wk and then energy restricted and fed a high-protein diet containing caseins, milk-soluble proteins (MSP), or a casein– MSP mixture (n ¼ 9 per group) as the only source of protein for 3 wk. Food intake was spread over 12 h, whereas in a previous experiment rats consumed their daily ration within 2 to 3 h. Weight and food intake were recorded. The body composition was measured by dual-energy x-ray absorptiometry before and after energy restriction. After 3 wk, the hind-limb muscles, the kidney, intestine, liver, and spleen weights, metabolic plasma parameters, and the liver and extensor digitorum longus muscle protein synthesis rates were measured in the postprandial state. Results: The food intake was similar in all groups. Energy restriction induced a significant decrease in body weight and fat mass (P < 0.001) and stopped the slow growth of lean body mass, with no differences between groups. Among all tissues, a significant effect was detected only for the intestine (P ¼ 0.0012), with a higher weight in the casein group. Postprandial liver and muscle protein synthesis rates were not different between groups. Conclusion: When using a high-protein diet spread over 12 h, the nature of the protein intake has no influence on the sparing of lean body mass during energy restriction in young overweight rats. Ó 2012 Elsevier Inc. All rights reserved. Introduction Obesity has reached epidemic proportions: more than 1 billion adults are overweight, and at least 300 million are clinically obese [1]. Obesity is a risk factor for cardiovascular diseases and diabetes and contributes strongly to the global burden of the associated health costs. Obese individuals seeking weight loss often use restrictive diets, which lead to a decrease in adiposity but also to a loss of fat-free mass [2]. This loss of fat-free mass and in particular muscle mass should be prevented because muscle is an emergency store of amino acids that can be used during stresses, allowing an organism to maintain homeostasis. This loss can be limited by including a sufficient amount of protein in the energy-restricted diet [3]. Previously we compared the capacity of caseins (slowly digested milk proteins) with that of milk-soluble proteins (MSP; rapidly digested leucine-rich proteins) to maintain lean body mass in overweight, energy-restricted rats [4]. In the present study, we investigated whether the timing of the intake of these proteins could have an influence on the sparing of lean body mass. Indeed, in our previous experiment [4], rats consumed their daily ration within 2 to 3 h. We found that, although the regulations of liver and muscle protein metabolisms were not the same, the final nitrogen balance (and thus whole-body protein mass) was not different between groups. In that experiment, postabsorptive muscle protein synthesis rates were higher in the casein-fed group than in the MSP-fed groups [4]. Given the results obtained in test-meal studies in humans [5–7], we postulated that the muscle protein balance (i.e., protein synthesis