Ultrasound is an inexpensive and practical alternative to gold standard measures of muscle mass. Changes in body position may lead to intra-muscular fluid shifts that may affect the reliability of ultrasound measures. We assessed test-retest reliability of ultrasound measures of muscle thickness and the effect of position on these measures. Measures of muscle thickness were made from B-mode ultrasound images in 18 healthy participants. Repeated measurements were made by one examiner from two participant positions; standing and recumbent, from 18 anatomical sites. Results demonstrated high test-retest reliability for measures taken in both participant positions [intra-class coefficient (ICC) scores 0.65 to 0.94], except for the recumbent posterior lower leg measures (ICC 0.34). Recumbent measures were significantly smaller than those taken with participants standing. Length of time participants spent lying down did not significantly affect measures, indicating that any changes in intra-muscular fluid related to the time spent recumbent are negligible.