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Clinical small bowel bacterial overgrowth (SBBO) syndrome can be objectified by bacterial overgrowth tests. As direct culture of jejunal aspirates has disadvantages, noninvasive tests such as breath tests (BTs) are used. Major drawback of lactulose BT might be rapid lactulose transit to the colon. We evaluated diagnosing bacterial overgrowth using experimental and standard BT, and culture and molecular-based methods.Bacterial overgrowth was analyzed in 11 controls and 15 SBBO predisposed subjects. During experimental breath testing, an occlusive balloon limited lactulose to the small intestine. Jejunal fluid was analyzed using culture and molecular-based methods. Bacterial overgrowth was diagnosed on the basis of 20 ppm hydrogen or methane increase above baseline within 90 minutes or more than 103 CFU/mL excluding lactobacilli and streptococci and furthermore using all published definitions.Experimental and standard BT showed no changes in timing of hydrogen excretion between controls and SBBO subjects. Using standard BT, 3/11 controls and 8/15 SBBO subjects were bacterial overgrowth positive. Total counts showed no significant differences between controls and SBBO subjects using culture and molecular-based methods. Bacterial overgrowth was diagnosed in 0/9 controls and 4/12 SBBO subjects using culture-based methods. Other definitions used in literature revealed no significant differences between controls and SBBO subjects.In a small group of subjects, the experimental BT did not improve the ability of lactulose BT to diagnose bacterial overgrowth. Culturing showed less bacterial overgrowth in controls compared with BT. Remarkably, current diagnostic criteria do not seem to be accurate in discriminating between SBBO subjects and controls.