The major microbial fermentation of forages and production of volatile fatty acids (VFA) takes place in the medial part of the rumen, whereas the absorption of VFA occurs through the rumen epithelium, for example the ventral sac. The objective was to study effects of forage neutral detergent fibre (NDF) content and time after feeding on the medial to ventral VFA and pH gradient as well as rumen motility in the rumen of heifers fed grass/clover silages. Four silages were harvested at different growth stages with NDF contents of 31–45% of DM and in vitro organic matter digestibilities of 75–82% and fed to four rumen-fistulated Jersey heifers at 90% of ad libitum level in a Latin square design, with half the ration fed at 0800 hours and 1530 hours. Rumen fluid was sampled hourly from 0730 hours to 1530 hours in the medial and ventral rumen, and analysed for pH and concentrations of VFA, L-lactic acid, and ammonia to assess ruminal chemical gradient. Reticular contractions were continuously recorded by a pressure transducer. Time relative to feeding affected rumen parameters as pH was generally lower and VFA content greater in medial compared with ventral rumen fluid. Greater NDF content of the silage caused lower VFA concentration and higher pH in the rumen mat, and therefore the gradient diminished at greater NDF content in the silages; an effect probably caused by reduced organic matter digestibility rather than digesta NDF properties. This study therefore suggests that VFA production decreased with greater NDF content of forages, whereas intra-ruminal equilibration increased.