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Health effects of whole grain foods are becoming more evident. In this study, we analysed the short-chain fatty acid profiles in urine and serum derived from the colonic fermentation process of 13C-barley meals, prepared from barley grown under 13CO2 atmosphere.In a crossover study, five volunteers ingested intact barley kernels (high content of non-starch polysaccharides (NSP) and resistant starch (RS)) and barley porridge (high content of NSP only). Using a newly developed stable isotope technology, we monitored 14 and 24 h postprandially 13C-acetate, 13C-propionate and 13C-butyrate in plasma and urine, respectively. The oro-cecal transit time (OCTT) of the meals was measured with the hydrogen breath test.The OCTT was 6 h and did not differ between the two test meals. An increase of 13C-acetate was observed already early after ingestion of the meals (<6 h) and was attributed to early fermentation of the test meal. A rise in plasma 13C-propionate in the fermentation phase could only be detected after the porridge and not after the kernel meal. An increase in 13C-butyrate was only found in the fermentation phase and was higher after the barley kernels. Urine 13C-short-chain fatty acids data were consistent with these observations.The difference in the profiles of 13C-acetate, 13C-propionate and 13C-butyrate indicates that NSP combined with RS results in an altered fermentation profile than dietary fibre alone.