Excess weight and obesity are often associated with ectopic adipose tissue accumulation in skeletal muscles. Intermuscular adipose tissue (IMAT) impairs muscle quality and reduces insulin-stimulated skeletal muscle glucose uptake. Although energy restriction and high protein intake can decrease IMAT, the effects and mechanisms of protein intake from an energy-restricted diet on protein and fat masses in skeletal muscle have received little attention. After establishing a diet-induced overweight and obese Sprague-Dawley rat model (half male and half female), rats were divided into five groups: normal control (NC; normal weight, general maintenance diet), model control (MC; overweight and obesity, high-fat diet), energy-restricted low protein (LP; overweight and obesity, 60% energy intake of NC, general maintenance diet), energy-restricted normal protein (NP; overweight and obesity, 60% energy intake of NC, high-protein diet 1), and energy-restricted high protein (HP; overweight and obesity, 60% energy intake of NC, high-protein diet 2). After 8 weeks, plasma and skeletal muscle (quadriceps femoris and gastrocnemius) samples were collected. Plasma levels of glucose, triglycerides, and hormones were analyzed, while contents of protein, fat, and factors associated with their synthesis and degradation were evaluated in skeletal muscles. Plasma concentrations of hormones contrasted protein and fat contents in skeletal muscles. Fat weights and contents of quadriceps femoris and gastrocnemius muscles in the NP group were significantly lower compared with LP and HP groups (P < 0.05). Moreover, concentrations of factors associated with the degradation of muscle fat were significantly higher in the NP group compared with LP and HP groups (P < 0.05). During energy restriction, protein intake equal to that of a normal protein diet increased lipolysis of quadriceps femoris and gastrocnemius muscles in rats of both sexes.