To investigate the regional gravity-dependent impact of mechanical ventilation and fluid overload on lung extracellular matrix (ECM) in healthy lungs. The glycosaminoglycans (GAGs) composition of the ventral and dorsal lung parenchyma was determined in anesthetized supine healthy rats mechanically ventilated for 4 hours in air: (a) at low (∼7.5 mL/kg) or high (∼ 23 mL /kg) tidal volume (V(T)) and 0 cmH2O positive end-expiratory pressure (PEEP); (b) at low or high V(T) at 5 cmH2O PEEP and (c) with or without 7 mL /(kg·h) intravenous saline infusion. Mechanical ventilation degraded lung ECM, with alveolar septa thinning and structural GAGs disorganization. Low V(T) ventilation was associated with significant tissue structure changes in both ventral and dorsal lung regions, while high VT mainly affected the dependent ones. PEEP decreased ECM injury mainly in the ventral lung regions, although it did not prevent matrix fragmentation and washout at high V(T). Intravascular fluid load increased lung damage prevalently in the ventral lung regions. Mechanical ventilation and fluid load may cause additive injuries in healthy lungs, mainly in ventral regions.