Dietary fibre concentration and particle size influence chewing behaviour, ruminal fermentation and digesta passage in dairy cows and through this, may impact nitrogen (N) use and excretion by the animals. The aim was thus to evaluate the effects of physically effective neutral detergent fibre (peNDF) concentration on chewing behaviour, ruminal fermentation, passage rate and protein metabolism in four lactating, rumen-cannulated Holstein cows in a 4 × 4 latin square design. Four total mixed rations with identical ingredients, chemical composition and a negative ruminal N balance (–2.1 g/kg dry matter) were tested. They varied in peNDF concentration, adjusted by feed mixing time: low (L), medium-low (ML), medium-high (MH) and high (H) peNDF. Nutrient intakes, number of total chews, organic matter digestibility and yield and efficiency of ruminal microbial protein synthesis responded quadratically to increasing peNDF concentration, with greater values for MH and ML diets. While rumination and total chewing intensity (min/kg dry matter) increased with increasing peNDF concentration, milk yield and composition, digesta passage rates and concentrations of ammonium-N and volatile fatty acids in rumen fluid were similar across diets. Energy-corrected milk yield and partitioning of N between milk and urine responded quadratically to increased peNDF concentration. Energy-corrected milk yield and the percentage of ingested N secreted via milk were lower, but the percentage of N intake excreted via urine was greater for MH and ML diets. Hence, feeding dairy cows a low-protein diet with varying peNDF concentrations affects their chewing behaviour, nutrient digestion and protein metabolism.