It has been suggested that the positive position of the O'Brien test (shoulder flexion, horizontal adduction, and internal rotation) tensions the bicipital labral complex relative to the negative position (shoulder flexion, horizontal adduction, and external rotation). This study measured active and passive tension in the long head of biceps in the 2 positions of the O'Brien test to validate the proposed anatomic basis of the test. Active tension was measured using surface electromyography of the long head of biceps in 12 healthy young adults. Passive tension was measured using a load cell attached to the intracapsular tendon of the long head of biceps in 5 cadaver specimens. Active and passive tension were higher in the negative position, thus refuting the proposed anatomic basis of the test. Although there may be an alternative basis for the test, the failure to support the proposed anatomic basis may partly explain the variable likelihood ratios obtained in clinical accuracy studies of the O'Brien test. The results of this study reinforce the need for anatomic validation during the development of clinical testing procedures.