ABSTRACTStress-induced abdominal dysfunction is an attractive target for probiotics. To investigate the effects of the probioticLactobacillus caseistrain Shirota on abdominal dysfunction, a double-blind, placebo-controlled trial was conducted with healthy medical students undertaking an authorized nationwide examination for academic advancement. For 8 weeks, until the day before the examination, 23 and 24 subjects consumed anL. caseistrain Shirota-fermented milk and a placebo milk daily, respectively. In addition to assessments of abdominal symptoms, psychophysical state, and salivary stress markers, gene expression changes in peripheral blood leukocytes and composition of the gut microbiota were analyzed using DNA microarray analysis and 16S rRNA gene amplicon sequence analysis, respectively, before and after the intervention. Stress-induced increases in a visual analog scale measuring feelings of stress, the total score of abdominal dysfunction, and the number of genes with changes in expression of more than 2-fold in leukocytes were significantly suppressed in theL. caseistrain Shirota group compared with those in the placebo group. A significant increase in salivary cortisol levels before the examination was observed only in the placebo group. The administration ofL. caseistrain Shirota, but not placebo, significantly reduced gastrointestinal symptoms. Moreover, 16S rRNA gene amplicon sequencing demonstrated that theL. caseistrain Shirota group had significantly higher numbers of species, a marker of the alpha-diversity index, in their gut microbiota and a significantly lower percentage ofBacteroidaceaethan the placebo group. Our findings indicate that the daily consumption of probiotics, such asL. caseistrain Shirota, preserves the diversity of the gut microbiota and may relieve stress-associated responses of abdominal dysfunction in healthy subjects exposed to stressful situations.IMPORTANCEA novel clinical trial was conducted with healthy medical students under examination stress conditions. It was demonstrated that the daily consumption of lactic acid bacteria provided health benefits to prevent the onset of stress-associated abdominal symptoms and a good change of gut microbiota in healthy medical students.