In this study, we aimed to determine if electromyography (EMG) normalization to maximal voluntary isometric contractions (MVIC) was influenced by subacromial pain in patients with subacromial impingement syndrome. Patients performed MVICs in unique testing positions for each shoulder muscle tested before and after subacromial injection of local anesthetic. In addition to collection of MVIC data, EMG data during an arm elevation task were recorded before and after injection. From a visual analog pain scale, patients had a 64% decrease in pain following the injection. Significant increases in MVICs were noted in 4 of the 7 shoulder muscles tested: anterior, middle and posterior deltoid, and lower trapezius. No significant differences were noticed for the upper trapezius, latissimus dorsi, or serratus anterior. MVIC condition (pre and post injection) had a significant influence on EMG normalization for the anterior deltoid and lower trapezius muscle. Results indicate that subacromial pain can influence shoulder muscle activity, especially for the deltoid muscles and lower trapezius. In addition, normalization to MVIC in the presence of pain can have unpredictable results. Caution should be taken when normalizing EMG data to MVIC in the presence of pain.