Background: Single subject trials offer an alternative approach to identify and characterize responders to a specific treatment. Aim: To test a new single subject trials design, called random starting day trial, to identify acid-related symptoms in dyspepsia. Methods: A total of 119 patients with functional dyspepsia entered a 12-day, double-blind random starting day trial. All patients started on placebo and switched to omeprazole 80 mg/day at a randomized and blinded day between day 5 and day 9, with active treatment continuing for the rest of the trial. Based on changes of a daily symptom score, response was defined as a sustained ≥50% reduction of symptoms within 3 days of active treatment. Results: Thirteen of 119 patients (11%) were classified as spontaneous responders because of complete symptom relief before switching to omeprazole. Of the remaining 106 patients, 15 (15.6%) were classified as responders. Five of six (83%) responders compared with 28 of 53 (53%) non-responders had pathological reflux. Multivariate testing identified symptoms suggestive of gastro-oesophageal reflux predictive of response. Conclusions: The random starting day trial design could identify a subset of dyspeptic patients with a uniform symptomatic response to acid-suppressive therapy. Response seems to be associated with gastro-oesophageal reflux. The random starting day trial needs to be further validated to be considered as a reliable instrument in clinical research.