Background: Obesity is associated with disrupted energy metabolism and skeletal muscle dysfunction. Dietary protein is important for the preservation of skeletal muscle mass and strength. Objectives: The objectives of this preliminary study were: 1) to assess the association of dietary protein intake with body composition, and muscle function; 2) to examine weather protein distribution pattern throughout the day are associated with body composition and substrate oxidation; and 3) to explore the association between body composition with substrate oxidation and energy expenditure in males and females with severe obesity. Methods: This cross-sectional study included 17 male and female bariatric surgery candidates. Dietary data, including protein intake was collected using a 3-day food journal and 24-hour food recall. Body composition was assessed using dual-energy X-ray absorptiometry. Substrate oxidation and resting energy expenditure were measured by indirect calorimetry. Muscle function was assessed by handgrip strength and 6-minute walk test. Results: Mean age and BMI were 43.9 ± 7.8 y and 46.9 ± 4.9 kg/m2, respectively. Daily protein distribution was uneven and had a skewness toward dinner. Males who ate ≥ 20 g protein at each meal had greater ALM in comparison to those who did not (38.0 ± 0.9 vs 35.2 ± 0.7, P = 0.03). ALM was an independent predictor of REE and fat oxidation rate. Females had altered substrate metabolism and decreased rates of fat oxidation. Conclusion: This study provided preliminary results that will help improve study design and considerations in the continued recruitment of participants.