Acid reflux episodes that extend to the proximal esophagus are more likely to be perceived. This suggests that the proximal esophagus is more sensitive to acid than the distal esophagus, which could be caused by impaired mucosal integrity. Our aim was to explore sensitivity to acid and mucosal integrity in different segments of the esophagus. A prospective study including 12 patients with gastroesophageal reflux disease. Two procedures were performed: an acid perfusion test and an upper endoscopy with electrical tissue impedance spectroscopy and esophageal biopsies. Proximal and distal acid sensitivity and tissue impedance were measured in vivo, and mucosal integrity at different esophageal levels was measured in vitro. Mean lag time to heartburn perception was shorter after proximal acid perfusion (0.8 minutes) than after distal acid perfusion (3.9 minutes); p = 0.02. Median in vivo tissue impedance was significantly lower in the distal esophagus (4563 •m) compared to the proximal esophagus (8170 •m); p = 0.002. Transepithelial permeability, measured by the median fluorescein flux was significantly higher in the distal (2051 nmol/cm²/h) than the proximal segment (368 nmol/cm²/h); p = 0.033. Intercellular space ratio was not significantly different between the proximal and distal esophagus. Conclusion In GERD patients off acid secretion-inhibiting medication, acid exposure in the proximal segment of the esophagus provokes symptoms earlier than acid exposure in the distal esophagus, whereas mucosal integrity is impaired more in the distal esophagus. These findings indicate that the enhanced sensitivity to proximal reflux episodes is not explained by increased mucosal permeability.