Protoporphyrin IX-triplet state lifetime technique (PpIX-TSLT) is a method used to measure oxygen (PO2 ) in human cells. The aim of this study was to assess the technical feasibility and safety of measuring oxygen-dependent delayed fluorescence of 5-aminolevulinic acid (ALA)-induced PpIX during upper gastrointestinal (GI) endoscopy. Endoscopic delayed fluorescence measurements were performed 4 hours after oral administration of ALA in healthy volunteers. The ALA dose administered was 0, 1, 5 or 20 mg/kg. Measurements were performed at three mucosal spots in the gastric antrum, duodenal bulb and descending duodenum with the catheter above the mucosa and while applying pressure to induce local ischemia and monitor mitochondrial respiration. During two endoscopies, measurements were performed both before and after intravenous administration of butylscopolamine. Delayed fluorescence measurements were successfully performed during all 10 upper GI endoscopies. ALA dose of 5 mg/kg showed adequate signal-to-noise ratio (SNR) values >20 without side effects. All pressure measurements showed significant prolongation of delayed fluorescence lifetime compared to measurements performed without pressure (P < .001). Measurements before and after administration of butylscopolamine did not differ significantly in the duodenal bulb and descending duodenum. Measurements of oxygen-dependent delayed fluorescence of ALA-induced PpIX in the GI tract during upper GI endoscopy are technically feasible and safe. © 2019 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.