Objectives To evaluate the effect of isometric shoulder extension in 90° shoulder flexion on the acromio-humeral distance, to establish the force required to achieve a clinically important increase in the acromio-humeral distance, and to investigate the practicality and reliability of real-time ultrasound measurement of the acromio-humeral distance in 90° shoulder forward flexion. Design Prospective single-group intervention. Setting King's College London, Guy's Campus. Participants Twenty healthy volunteers [five males and 15 females (40 shoulders)] with a mean age of 32 (standard deviation 10, range 19 to 55) years were recruited from the faculty and staff at King's College London. Interventions The acromio-humeral distance in asymptomatic participants was measured using real-time ultrasound in the neutral position at rest, at 90° shoulder flexion at rest, and while performing an isometric pull-down exercise at 100%, 50%, 30% and 10% maximal voluntary isometric contraction. Main outcome measures Real-time ultrasound measures of the acromio-humeral distance. Results Of the 20 participants, 38 shoulders were imaged. In 90° shoulder flexion, pull-down exercises at all levels of force increased the acromio-humeral distance compared with no pull-down (P < 0.05), but this was only clinically significant in males. Measures had excellent short-term intra-operator reliability. Conclusions Isometric pull-down exercises lead to an increase in the acromio-humeral distance in asymptomatic males that may be clinically important, and therefore may be an appropriate exercise for patients with shoulder pathology. Ultrasound measurement of the acromio-humeral distance in 90° shoulder flexion is practical and reliable.