The aim of this study was to investigate the microbial protein yield of different pure carbohydrates to contribute to a more precise prediction of the microbial protein formed in the rumen. In a first experiment, sucrose, wheat starch, microcrystalline cellulose and citrus pectin were incubated for 8 and 24 hr in the modified Hohenheim gas test (HGT) system (3 runs × 2 syringes) including gas production, ammonia and short-chain fatty acid concentration measurements. Ammonia values were used for estimation of the microbial protein formation. In a second experiment, the same substrates were incubated for 96 hr in the HGT system (2 runs × 3 syringes) and gas production was measured after 2, 4, 6, 8, 12, 16, 24, 30, 36, 48, 60, 72 and 96 hr of incubation to obtain the fermentation kinetics and the time of half-maximal gas production (t1/2 ) of the substrates. The substrates differed considerably in their fermentation kinetics, and therefore, comparison on the basis of t1/2 was chosen as the most meaningful. At t1/2 , microbial protein yield [g/kg dry matter] was higher for cellulose than for sucrose and pectin and higher for starch than for sucrose. The microbial protein expressed in g/L gas production was higher for starch and cellulose than for sucrose and pectin at t1/2 . Effects of carbohydrates related to ruminal pH may remain undetected in in vitro trials. © 2019 Blackwell Verlag GmbH.