Elevated levels of both hydrogen and molar proportions of propionate to acetate and butyrate are found in the bovine rumen right after meals. Hydrogen is believed to thermodynamically control fermentation pathways in the rumen in favor of propionate production. Elevated levels of hydrogen inhibit reoxidation of NADH to NAD⁺. Propionate production, however, enables this reoxidation and explains why elevated levels of hydrogen and propionate are hypothesized to occur simultaneously. Nonetheless, hydrogen partial pressure in the rumen headspace may not be high enough to inhibit NADH reoxidation. Furthermore, studies reporting diurnal patterns of volatile fatty acids (VFA) concentration and hydrogen pressure simultaneously, if any, are limited. The aim of this study is to monitor daily patterns of hydrogen pressure, pH, and VFA concentration in the bovine rumen and to calculate thermodynamic inhibition of specific fermentation pathways. Four rumen fistulated multiparous lactating cows were used in a crossover design with 2 17-d experimental periods and a control and treatment diet. Both diets consisted of 40% corn silage, 30% grass silage and 30% concentrates on DM basis. The treatment diet had a 2.5% higher fat content by supplementing the concentrate with linseed oil. On d 11, rumen headspace gas and fluid samples were taken at 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10 h after morning feeding using a custom fistula lid enabling rumen gas and fluid sampling. Gas samples were analyzed for hydrogen pressure, and fluid samples for pH and concentrations of dissolved hydrogen as well as VFA. Fluid samples were also taken for transcriptome analysis to monitor microbiome gene expression. From d 13 to 17 cows were housed in respiration chambers to relate rumen headspace pressure to emissions of hydrogen. An increase of hydrogen partial pressure by 2 orders of magnitude up to 30 mbar was observed following feeding. This finding might indicate hydrogen partial pressure to be high enough to inhibit NADH reoxidation.