The abdominal microsurgical flap based on the deep inferior epigastric artery perforator (DIEP) flap has become the most popular option worldwide for autologous breast reconstruction. Several authors have investigated the results of reconstructed breasts, but the literature lacks systematic reviews exploring the donor site of the abdominal wall. To fulfil our aims, a new diagnostic muscle imaging analysis was designed and implemented. This study focused on rectus abdominal muscle morphology and function in a single series of 12 consecutive patients analysed before and after breast reconstruction with a microsurgical DIEP flap. Patients were divided into two groups, namely, “ipsilateral reconstruction” and “contralateral reconstruction”, depending on the side of the flap harvest and breast reconstruction, then evaluated by computed tomography (CT) scans scheduled for tumor staging, and clinically examined by a physiatrist. Numerous alterations in muscle physiology were observed due to surgical dissection of perforator vessels, and rectus muscle distress without functional impairment was a common result. Postoperatively, patients undergoing “contralateral reconstruction” appeared to exhibit fewer rectus muscle alterations. Overall, only three patients were impacted by a long-term deterioration in their quality of life. On the basis of the newly developed and implemented diagnostic approach, we concluded that DIEP microsurgical breast reconstruction is a safe procedure without major complications at the donor site, even if long-term alterations of the rectus muscle are a common finding.