Subacute ruminal acidosis (SARA) continues to be a common and costly metabolic disorder in high-producing dairy cows worldwide. The objective of this study was to evaluate if increasing the concentration of physically effective neutral detergent fiber (peNDF) in diets can reduce the risk of SARA in cows fed a high-concentrate diet. Thirty second-parity Holstein cows in mid lactation (131 ± 8.3 d in milk) were randomly allocated to 3 dietary treatments (10 dairy cows per group): high (11.3%, high peNDF_8.0), medium (10.6%, medium peNDF_8.0), or low (9.0%, low peNDF_8.0) concentration of peNDF_8.0. The diets were prepared by mixing the same total mixed ration (57% concentrate and 43% roughages) for 10, 18, or 60 min, respectively. The treatments were fed for 36 d with 21 d for adaptation and 15 d for sampling. The peNDF_8.0 intake was positively correlated with the peNDF_8.0 concentration. Chewing and ruminating times adjusted for dry matter intake and NDF intake were linearly increased with the increased dietary peNDF_8.0 concentration. The high peNDF_8.0 diet decreased the number of meals per day. The increased dietary peNDF_8.0 concentration linearly increased the rumen fluid pH, the molar percentage of acetate and isobutyrate, acetate-to-propionate ratio, and ammonia nitrogen concentration, but linearly decreased the molar percentages of propionate and valerate. The total VFA concentration and the molar percentages of butyrate and isovalerate remained unchanged. Meanwhile, the increase in the peNDF_8.0 concentration of the diet linearly increased the activities of carboxymethyl cellulase, avicelase, β-glucanase, and ferulic acid esterase in rumen fluid, but did not affect the activities of xylanase. Total plasma antioxidant capacity, γ-glutamyl transpeptidase activity, and plasma concentrations of total protein, albumin, creatinine, and malondialdehyde were linearly decreased by the increased dietary peNDF_8.0 concentration. The increase in peNDF_8.0 concentration raised the plasma concentrations of glucose, triglyceride, cholesterol, and blood urea nitrogen. Somatic cell counts in the milk were positively correlated with the dietary peNDF_8.0 concentration. The feed and milk energy efficiencies were unaffected by the treatments. Shortening the total mixed ration mixing time may be a practical strategy to increase the peNDF_8.0 concentration and reduce the risk of SARA in dairy cows fed high-concentrate diets.

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