Mammography has become widespread as a standard method for breast cancer screening. For women undergoing mammography, compression of the breast and special positioning are requested, and they cause the physical burden and pain. However, the reality of the physical burden and subjective pain associated with mammography are not fully understood. We therefore measured the muscle activity of subjects during positioning for mammography screening using surface electromyography to evaluate the physical burden and pain associated with the positioning. The subjects consisted of 15 women (age: 44.4 ± 6.56 years old, height: 160 ± 6.7 cm, weight: 55.08 ± 3.94 kg, and body mass index: 21.4 ± 2.21). Measurements were performed in the mediolateral oblique position, a standard position for mammography. The target muscles were the sternocleidomastoid, biceps, trapezius and gastrocnemius muscles. A portable multi-purpose bio-amplifier was used for the measurements. Visual analogue scale (VAS), which is a tool for self-assessment of subjective pain, was used for pain measurement. The analysis of variance showed the significant difference in the amounts of muscle activities in all the target muscles between the relaxation phase before mammography positioning and the stress phase during mammography positioning. The sites with the increased muscle activities were consistent with the sites of pain measured with the VAS. These results suggest that positioning during mammography affects the muscle activity and that the increased muscle activity could be related to the pain. Understanding muscle activities during mammography is invaluable in making the pain reduction program for the subjects undergoing mammography.