BACKGROUND: Quantitative neuromuscular ultrasound is increasingly used to study muscle pathology and changes in muscle mass during critical illness. Advantages of ultrasound are high axial resolution, low procedural risks, no ionizing radiation, and ease of use early in the course of disease. However, ultrasound is known to be an operator dependent imaging modality and the intensive care unit setting poses additional challenges to obtaining reliable measurements. There is limited evidence validating the feasibility and reliability of its application in this setting. OBJECTIVE: To conduct a standardized protocol for measuring muscle linear depth and cross-sectional area in critically ill populations with a high degree of interrater agreement and feasibility. DESIGN: Prospective observational cohort study of interrater reliability. SETTING: Medical intensive care unit at an academic medical center and a level one trauma and burn center. PATIENTS: Fifteen critically ill patients were evaluated using a standardized ultrasound protocol measuring total elbow flexor, knee extensor, and tibialis anterior depth, as well as rectus femoris cross-sectional area. Each site was independently scanned by two investigators. Reliability of measurements between observers was determined by calculating intraclass correlation coefficients (ICCs) using a two-way random effects model and absolute agreement. An ICC > 0.75 was considered good and >0.90 was considered excellent. RESULTS: In critically ill patients, interrater reliability of linear depth measured at elbow flexor, knee extensor, tibialis anterior, and cross-sectional area of rectus femoris sites was good to excellent with ICC between 0.87 (0.54-0.97) and 0.99 (0.97-1.00). Interrater reliability was improved by creating a summary index of measures resulting in an ICC of 0.99 (0.98-1.00). Feasibility, as definite by the percentage of each measure that was obtainable, ranged from 75%-100%. CONCLUSIONS: Using a standardized protocol, ultrasound measures obtained in critically ill patients demonstrated high levels of interrater agreement with good to excellent feasibility.