Background:13C-hiolein is a purified algal triglyceride which is synthesized with 98% of all carbon atoms of the molecule uniformly 13C-labeled. Orlistat is the new name for tetrahydrolipstatin (THL), which is a specific and potent inhibitor of gastrointestinal lipases which induces artificial fat malabsorption in humans. The aim of the present study was to use the 13C-hiolein breath test (HBT) to assess THL-induced fat malabsorption in healthy volunteers. Methods: Eight healthy volunteers of normal body weight underwent 2 study periods of 4 days of diet with or without THL 120 mg t.i.d. On day 5, a HBT was performed. 13CO2 recovery in breath samples was measured over 24 h by isotope ratio mass spectrometry. Results: The peak 13CO2 excretion occurred only after 5 h in both treatment groups with little difference during the first 4 h between the groups. THL potently reduced fat digestion and absorption with the most pronounced effect observed after 8 h: 1.1± 0.2 vs. 2.3 ± 0.3% dose in control experiments (p < 0.05). The 24-hour cumulative 13CO2 excretion was also significantly reduced by THL: 14.9 ± 2.2 vs. 28.4 ± 4.1% dose in control experiments (p < 0.05). In 6 of the 8 subjects, the cumulative 13CO2 excretion was lower with THL 120 mg t.i.d. than with placebo. Conclusion: The HBT well reflects THL-induced artificial fat maldigestion in healthy volunteers.