Exercises for lower trapezius (LT) often use overhead positions, causing compressive forces to the subacromial space. Scapular retraction would be an alternative to activate LT muscle. The present study aimed to assess the excitation levels of infraspinatus, upper trapezius, and lower trapezius muscles during a scapular retraction exercise under progressive adduction loads in subjects with and without painful shoulder. Electromyography of infraspinatus (IS), upper trapezius (UT), and LT was recorded during scapular retraction under progressive adduction loads of 42 participants, divided into two groups: with (SP, n = 26) and without shoulder pain (nSP, n = 16). The adduction loads of 20%, 30%, 40%, and 50% of the maximal voluntary contraction (MVC) were applied using a load cell. Normalized electromyography and the ratio between UT and LT (UT:LT) were used for statistical analysis. No differences were observed between groups, but a condition effect occurred for all muscles: UT showed higher values at 50% vs. 20% of MVC (p = 0.004); LT showed higher values on 40% and 50% of MVC (p = 0.001; 0.006). Higher values for IS were noted at 40% of MVC (vs. 20% of MVC; p = 0.04) and at 50% of MVC (vs. 20% of MVC; p = 0.001, vs. 30% of MVC, p = 0.001; vs. 40% of MVC; p = 0.001). UT:LT showed lower values at 50% of MVC (vs. 20% of MVC; p = 0.001 and vs. 30% of MVC; p = 0.016). Scapular retraction with adduction loads at 40–50% is an alternative to overhead exercises aiming to activate the LT and the IS muscles. The exercise ensures higher levels of LT and IS excitation without increasing UT excitation.