This study investigated effects of experimentally-induced subacromial pain, induced via hypertonic saline injection, on shoulder muscles activity. Electromyographic activity of 20 healthy participants was assessed for humeral elevation and descent for the control and experimental pain conditions, using fine wire electrodes for subscapularis and supraspinatus and surface electrodes for middle deltoid, upper trapezius, lower trapezius, infraspinatus, and serratus anterior. Normalized mean amplitudes were analyzed for each muscle for four phases for elevation and descent, respectively. Repeated measures analysis of variances (ANOVAs) were used to determine differences between muscle activity in the control and experimental condition for the four phases of elevation and descent. Differences for mean normalized amplitudes were not significant during humeral elevation. Increased activity was found for the pain condition for serratus anterior and middle deltoid during the first (120-90°) and third (60-30°) parts and decreased activity for infraspinatus in the second half of descent (60-0°). No significant differences were found during descent for upper and lower trapezius, subscapularis and supraspinatus. While increased serratus anterior activity during 60-30° of descent may be protective, increased middle deltoid and decreased infraspinatus activity during the same range may threaten subacromial tissues in that range. Overall the changes in muscle activation were individual specific, particularly during the concentric elevation phase.