Background: Shoulder impingement syndrome, a major cause of shoulder pain, involves weakness of the scapular retractor muscles. The major scapular retractor muscles are the middle trapezius and rhomboid major muscles; however, the latter is excluded in most studies. Objects: We aimed to measure the thickness of the middle trapezius and rhomboid major muscles using an ultrasonic diagnostic imaging system while performing four different shoulder retraction exercises and comparing the thicknesses and ratio of the thicknesses of these muscles. Methods: The thickness of the middle trapezius and rhomboid major muscles was measured in 24 healthy adults using ultrasound. Muscle thickness was measured three times in the Reference posture and four times while performing four different exercises that involved scapular retraction. The averages and standard deviations of the measured muscle thicknesses were obtained and compared. The ratio of muscle thickness and rate of changes in muscle thickness between the reference posture and the four exercises were compared. Results: For both, male (n = 10) and female (n = 14), there was a significant difference in the thickness of the middle trapezius muscle between the reference posture and the four exercises (p < 0.05) and in the thickness of the middle trapezius and rhomboid major muscles between male and female (p < 0.05); however, there was no significant difference in the ratio of the thicknesses of these muscles. Although a significant difference in the rate of change in muscle thickness during the four exercises was noted, there was no significant difference in the ratio of change in muscle thickness. Conclusion: This study demonstrates the ratio of the thicknesses of the middle trapezius and rhomboid major muscles and the rate of change in their thickness during exercises involving scapular retraction in healthy people in their 20s–30s.