Background: Chronic post-surgical pain is a condition persisting at least three months after surgery. It is estimated that 25-60% of patients who underwent breast cancer removal surgery suffer from post-mastectomy pain syndrome and occurred anxiety, depression, sleep disturbance and catastrophizing. Physical activity can reduce the risk of chronic diseases and has a good impact on mood and cognitive function. Aim of this study was to evaluate the effects of physical activity on the intensity of pain, depression and anxiety in women underwent mastectomy for breast cancer removal. Methods: A prospective observational unicentric cohort study was performed. Patients were female underwent unilateral or bilateral mastectomy. Numerical Rating Scale (NRS), was used to assess pain intensity, Beck’s Depression Inventory (BDI) for depression and Generalized Anxiety Disorders-7 (GAD-7), for anxiety evaluation. Physical activity was assessed by International Physical activity questionnaire (IPAQ). Interleukin (IL)-17, IL-1β, cortisol, adrenocorticotropic hormone (ACTH) and brain-derived neurotrophic factor (BDNF) were also evaluated in the blood of patients. All the evaluation was assessed 3 and 6 months after the surgery. Results: Adequate physical activity reduced intensity of pain, depression and anxiety symptoms in women affected by post-mastectomy pain syndrome. Moreover, adequate active women, showed a reduction of biomarkers of inflammation, cortisol, ACTH and an increase of BDNF. Conclusions: Our results suggest that physical activity can improve quality of life, reducing intensity of pain, inflammatory markers and it can be useful in the reduction of associated anxiety and depression.