Background: Irritable bowel syndrome (IBS) patients often experience meal-related symptoms, which might be related to visceral hypersensitivity. The Lactulose Nutrient Challenge Test (LNCT) is a non-invasive measure of sensitivity in relation to a meal. We aimed to define a cut-off for hypersensitivity to LNCT based on results in healthy volunteers, and to characterize patients with LNCT hypersensitivity. Methods: IBS patients (n=273) and healthy volunteers (n=133) that completed LNCT were included. During LNCT, eight symptoms are rated on Likert scales and breath hydrogen/methane is assessed every 15min for 4h. Additional questionnaires assessed severity of GI, somatic and psychological symptoms. A subset completed oro-anal transit time and rectal sensitivity investigations. LNCT hypersensitivity was defined based on the average area under the curve of healthy volunteers (95th percentiles) for abdominal pain, bloating, and gas/flatulence. Statistics included univariate and logistic regression [OR (95% CI)] analyses. Results: In total, 76% of IBS patients were hypersensitive to LNCT. These patients had distinct characteristics, as they were more frequently female, reported more severe GI, non-GI somatic, and psychological symptoms, and had higher breath hydrogen production and lower rectal pain thresholds. More severe GI symptoms [2.05 (1.05–3.99)], lower rectal pain threshold [0.93 (0.88–0.99)], and higher breath hydrogen production [1.14 (1.02–1.29)] were identified as independent predictors of hypersensitivity to LNCT. Discussion: The LNCT is useful as a non-invasive and physiologic tool to test GI sensory function in relation to a meal. Moreover, overall GI symptom reporting, visceral hypersensitivity, and hydrogen production are important factors involved in postprandial symptoms in IBS.