Subacromial space is the margin between the humeral head and the acromion. People with subacromial space stenosis (SSS) caused by narrowing of the acromiohumeral distance (AHD) are predisposed to impingement syndrome or rotator cuff disease (RCD). Ultrasound (US) enables physicians or radiologists to dynamically assess AHD in different shoulder joint positions and has been well studied. However, a few studies have discussed and quantified the severity of SSS with the ratio of AHD displacement and the slope of AHD decease. The present study aims to investigate the dynamic change of AHD using a tracking algorithm optimized for tracking bone movement, and evaluate the test-retest reliability in healthy volunteers. Volunteers without history of recent trauma, shoulder fractures, shoulder instability, shoulder surgery or clinical treatment for a shoulder injury were recruited in this study. Dynamic US AHD measurement was performed by an experienced physician on all participants bilaterally using three views of B-mode image in passive movement of upper arm (from neutral position to 45 degrees abduction). The dynamic images were divided into frames and tracked with NIH software by another two observers without any information of subjects. (1) Maximal AHD; (2) minimal AHD; (3) the slope of linear function of AHD decrease were then analyzed to determine the intraclass correlation coefficient (ICC) for inter- and intra-observer reliability. 16 AHD measurements were performed on bilateral shoulders in 8 college volunteers (6 males, 2 females, age: 25 ± 1.3 years). Except the minimal AHD, most parameters demonstrated high ICC value (0.82 – 0.93) meaning good to excellent agreement for both inter- and intra-observer comparisons. Dynamic US with tracking technique could provide an appropriate quantitative measurement for AHD and give more clinical information in shoulder impingement or rotator cuff disease.