Chest optimization, an evidence-based protocol-guided multimodal chest physiotherapy consisting of body positioning, sputum mobilization, bronchodilation, and lung hyperinflation, may be routinely administered to ventilator-dependent patients with low cervical spinal cord injury (CSCI) for improving pulmonary functional outcomes that facilitate weaning from mechanical ventilation. We undertook this study to determine whether position-specific chest optimization was associated with changes in spontaneous breathing trial (SBT) duration. Cardiac output (CO), alveolar minute volume (MValv), carbon dioxide elimination (VCO(2)), and static chest compliance (Cst) were measured during chest optimization; then MValv and rapid shallow breathing index (RSBI) were measured during SBT. Study participants (N = 12) were clinically stable ventilator-dependent patients with low CSCI. Trendelenburg chest optimization (TCO) was associated with significant increases in SBT (p < 0.001), CO (p < 0.001), MValv (p < 0.003), VCO(2) (p < 0.001), and Cst (p < 0.002). SBT following TCO was associated with significant increases in MValv (p < 0.03) and RSBI (p < 0.002). These preliminary findings suggest the importance of proper recumbent body positioning during evidence-based, protocol-guided multimodal chest physiotherapy for ventilator-dependent patients with low CSCI.