The objective was to study the variation in the amino acid profile of microbial fractions obtained after feeding 16 total mixed rations for dairy cows in a Rusitec. Each ration was incubated for 15 d in 3-fold replicate. The rations showed high variation in the inclusion level of different ingredients and the content of proximal nutrients, fiber fractions, and energy. Three microbial fractions were isolated by centrifugation. The reference microbes (RM) were isolated from the liquid effluent of vessels between d 7 and 15 of incubation. Solid-associated microbes (SAM) were detached with methylcellulose from feed residues after incubation, and liquid-associated microbes (LAM) were obtained from the liquid content of the vessel. Both SAM and LAM were obtained only once for each vessel at the end of the incubation period. Across all rations, significant differences were found between RM, LAM, and SAM in amino acid concentration for some, but not all, amino acids. Within each microbial fraction, significant differences in the content of amino acids were found between rations. Multiple linear regression analysis did not show that the content of a certain nutrient or the inclusion rate of single feedstuffs could be used to predict the amino acid profile of microbial protein with an adequate level of accuracy. Further studies are necessary before the supply of individual microbial amino acids to the cows' duodenum can be modeled and predicted in dependence of diet data.