Ultrasonic measurement of the rectus femoris (RF) is a novel, proxy measure for muscle strength. The impact of hip flexion/head of bed positioning on RF cross-sectional area (CSA) has not been fully explored. This study describes and compares differences in RF CSA across four degrees of hip flexion. This repeated-measures, comparative study enrolled healthy, pre-menopausal women (n = 20). RF CSA of the dominant leg was measured using the SonoSite M-Turbo ultrasound system with the head of bed at 0°, 20°, 30°, and 60°. One-way repeated measures indicated significant differences in RF CSA, F(3, 17) = 14.18, p < .001, with variation in hip flexion/head of bed elevation and significant RF CSA differences between: (a) 0° and 20°, (b) 0° and 30°, (c) 0° and 60°, and (d) 20° and 60°. Standardizing patient positioning when conducting ultrasonic measurement of RF CSA is vital for researchers who assess muscle mass.