Refractory heartburn despite acid suppression may be explained by ongoing gastroesophageal reflux disease (GERD) or functional heartburn (FH), i.e., symptoms without evidence of GERD. Impedance-pH monitoring (impedance-pH) detects acid and nonacid reflux and is useful for evaluating acid-suppressed, refractory patients. Intercellular space diameter (ISD) of esophageal epithelium measured by transmission electron microscopy (TEM) is a marker of epithelial damage present in both erosive and nonerosive reflux disease. ISD has not been used to study refractory heartburn or FH. Our aim was to compare ISD in healthy controls and refractory heartburn patients with GERD and FH.In refractory heartburn patients (heartburn more than twice/week for at least 2 months despite proton pump inhibitor (PPI) b.i.d.), erosive esophagitis and/or abnormal impedance-pH (increased acid exposure or positive symptom index) defined GERD; normal esophagogastroduodenoscopy (EGD)/impedance-pH defined FH. Asymptomatic, healthy controls had normal EGD and pH-metry. Mean ISD in each subject, determined by blinded TEM of esophageal biopsies, was the average of 100 measurements (10 measurements in each of 10 micrographs).In all, 11 healthy controls, 11 FH, and 15 GERD patients were studied. Mean ISD was significantly higher in GERD compared with controls (0.87 vs. 0.32 μm, P=0.003) and FH (0.87 vs. 0.42 μm, P=0.012). Mean ISD was similar in FH and controls (0.42 vs. 0.32 μm, P=0.1). The proportion of patients with abnormal ISD was significantly higher for GERD compared with FH (60 vs. 9%, P=0.014).ISD is increased in refractory heartburn patients with GERD but not those with FH. Our findings suggest that measurement of ISD by TEM might be a useful tool to distinguish GERD from FH in patients with refractory heartburn.