Fructooligosaccharides (FOS) are nondigestible carbohydrates assumed beneficial because they stimulate the protective colonic microflora (bifidobacteria, lactobacilli) that produce organic acids that, in turn, increase host defence against invasive pathogens. However, studies show that FOS increases cytotoxicity of intestinal contents (fecal water), mucin excretion, and intestinal permeability in rats, reducing resistance to infection (since host defence depends on barrier function). This study aims to prove whether the adverse adverse effects of FOS that occurred before infection in rats would occur in humans. This is important because FOS has been added to a variety of products including dairy products and infant formulas. This is a double-blind, placebo-controlled crossover study design with 2 supplement periods of 2 wk. separated by 1 washout period of 2 wks. 34 healthy men were randomly divided in 2 groups. Subjects consumed either lemonade with 20g of FOS or 6g of sucrose (placebo) per day in 3 divided doses (morning, afternoon, and evening). They avoided dairy products and calcium rich foods (since FOS-induced adverse effects in rats is inhibited by calcium intake), foods high in fermentable nondigestible carbohydrates and pro- or prebiotics. Alcohol consumption was restricted. Habitual diet was otherwise maintained. The lemonade also contained the intestinal permeability marker chromium EDTA (CrEDTA). On the last 2 days of both supplement periods, quantitative food intake (self-reported) was measured, 24-h urine samples taken, and gastrointestinal symptoms rated (visual analogue scale). 24-h fecal samples were also collected. Dietary FOS consumption increased bifidobacteria, lactobacilli, lactic acid and decreased fecal pH. Cytotoxicity of fecal water and urinary and fecal CrEDTA excretion were not affected by FOS. Frequency of flatulence, bloating, abdominal pain and cramps were increased in the FOS period. The concept of stimulating endogenous microflora and intestinal organic acid production by rapid fermentation of nondigestible carbohydrates is beneficial for the intestinal barrier in humans is not supported.