Ionizing radiation as a cancer therapy is associated with a variety of undesirable side effects. Consequently, radiotherapy can negatively affect neuromuscular function. Clinical observations have identified problems with swallowing and voice function. Our study aims to evaluate the impact of radiotherapy on laryngeal soft tissues using image analysis to quantify its effect on the structure of the vocalis and thyroarytenoid muscles. Case control study, retrospective analysis. We collected total laryngectomy specimens from six patients with persistent or recurrent cancer who had received preoperative radiotherapy (60-66 Gy). The control group consisted of total laryngectomy specimens from six patients who underwent surgery as primary treatment. Sampling of the specimens only included non-cancerous laryngeal tissue. Laryngeal histological slices were evaluated using digital morphometric analysis system. Percentage of fibrosis and density of muscle fibers within the thyroarytenoid muscle were evaluated in both groups. We found no significant quantitative differences in muscle fibrosis (7.92% vs. 7.52%, P > 0.1). Changes were rather qualitative and included changes in the organization of the muscular fibers. A significant reduction in muscle fibers, however, was observed in the samples from irradiated larynges (66.45% vs. 42.03%, P < 0.01). Our analysis suggests that radiotherapy is responsible for a significant reduction in muscle fibers in the thyroarytenoid muscle and that these changes occur during treatment or relatively early after its completion. Loss of muscle mass after irradiation correlates with clinical observations of muscle weakness and decreased function in patients who undergo radiotherapy.