BACKGROUND: Maintaining sufficient dietary protein intake is important for nutritional status, muscle mass, and healthy aging, but the clinical effects of high protein diet remained controversial.. METHODS: This 12-week randomized controlled trial enrolled community-dwelling middle-aged and older adults to evaluate clinical effects of daily diet with different protein density.. All participants received 10 frozen meals per week for 12 weeks containing either 15% daily calorie of protein (regular-protein group, RPG), or 25% daily calorie of protein (high-protein group, HPG). Bioimpedance analysis was used to assess body composition, and the magnetic resonance imaging on both mid-thighs was performed to measure muscle mass and the intramuscular adiposity. RESULTS: This trial enrolled 70 participants, and data of 52 participants (mean age: 53.7 ± 8.3 years, 53.8% male; 25 in RPG and 27 in HPG) were available for analysis. Baseline demographic characteristics, functional assessment, body composition and muscle parameters, and laboratory data were similar between groups. During the study period, participants of both groups significantly reduced body weight, body mass index, total body fat percentage, and handgrip strength, but not in the relative appendicular muscle mass). Compared to participants of RPG, HPG participants showed significant improvement in 6-minute walking distance, increased high-sensitivity C-reactive protein and marginal reduction in intramuscular adiposity than RPG participants. CONCLUSIONS: Higher dietary protein intake significantly improved physical endurance and marginally reduced intramuscular adiposity, but increased the inflammatory biomarker among middle-aged and older adults. Further study is needed to explore long-term effects of high-protein diet among middle-aged and older adults..