Background: This study investigated whether diaphragmatic breathing intervention could lead to acute improvements in trunk and shoulder mobility and pulmonary function in healthy young adults. Methods: Twenty-six physically active males (aged 24.3 ± 2.0 years, body height of 182.9 ± 6.4 cm, and body weight of 82.8 ± 10.4 kg) were randomly assigned to either an experimental or a control group. The experimental group underwent a 22 min diaphragmatic breathing intervention in a lying position. The control group lay passively, breathing naturally. Mobility assessments (chest expansion, thoracic spine rotation, lateral trunk flexion, and shoulder girdle mobility) and pulmonary function tests (forced vital capacity, forced expiratory volume in one second and their ratio) were conducted before and after the intervention. Results: Only experimental group showed significant improvements after the intervention (p ≤ 0.01) in the chest expansion (+22.2%, ES = 0.62), thoracic spine rotation (+21.7%, ES = 0.76 on the left and +23.3%, ES = 0.84 on the right side), lateral trunk flexion (+11.7%, ES = 0.62 on the left and +15.4%, ES = 1 on the right side), shoulder girdle mobility (+20.2%, ES = 0.44 on the left and +21.5%, ES = 0.38 on the right side), forced vital capacity (+4.7%, ES = 0.39) and reduction (p ≤ 0.01) in ratio between forced expiratory volume in one second and forced vital capacity (−4.6%, ES = 0.47). Conclusion: The results revealed that a 22 min diaphragmatic breathing intervention could immediately improve trunk and shoulder mobility and pulmonary function, likely due to anatomical relationships and more efficient use of respiratory muscles, especially the diaphragm.