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We measured the effect of physically effective NDF (peNDF) in diets containing lower or higher concentrations of undegraded NDF at 240 h of in vitro fermentation (uNDF240) on DMI, milk production and composition, chewing behavior, ruminal responses, and nutrient digestibility. Sixteen Holstein cows (8 ruminally cannulated) averaging 123 ± 9 (SD) DIM were used in a replicated 4 × 4 Latin square design with 4-wk periods. Cows were fed TMR that differed in uNDF240 and peNDF content primarily by changing forage-to-concentrate ratio and particle size of timothy hay. Treatments were arranged as a 2 × 2 factorial arrangement consisting of (1) lower uNDF240 (LU; 8.9% of TMR DM) or higher uNDF240 (HU; 11.4% of TMR DM) and (2) lower peNDF (LP; 18.6%-20.1% of TMR DM) or higher peNDF (HP; 21.8%-22.0% of TMR DM). Physically effective uNDF240 (peuNDF240) was calculated as the product of the dietary pef (% of TMR DM retained on a ≥1.18-mm sieve with vertical dry sieving) and uNDF240 (as a percentage of DM) with the purpose of combining particle size and undegradability of fiber into one measure. Dietary peuNDF240 concentrations were 5.4% (LU-LP), 5.8% (LU-HP), 5.9% (HU-LP), and 7.1% (HU-HP) of TMR DM. Cows were housed in individual tiestalls, fed TMR once daily, and milked 3×/d. Data were analyzed as a factorial arrangement of treatments within a replicated Latin square design using the MIXED procedure of SAS (ver. 9.4; SAS Institute Inc., Cary, NC). The model included the fixed effects of uNDF240, peNDF, uNDF240 × peNDF, period within replicated square, and square. Cow within square was a random effect. There was an interaction between dietary peNDF and uNDF240, with cows fed the HU-HP diet consuming 10% less DM (24.9 kg/d) than cows fed the HU-LP (27.4 kg/d) or either LU diet (27.4 kg/d). Diets higher in peNDF content elicited greater peNDF intake. However, there was an interaction between uNDF240 and peNDF for uNDF240 intake driven by the HU-LP diet (3.1 kg/d) compared with the HU-HP (2.9 kg/d) and LU diets (2.4 kg/d). Intake of peuNDF240 reflected dietary pef and uNDF240 content, as expected. There was an effect of uNDF240 and peNDF on ECM production, and it tracked with dietary peuNDF240 (47.5, 45.2, 46.5, and 44.1 kg/d for diets LU-LP, LU-HP, HU-LP, and HU-HP, respectively). Mean ruminal pH increased, total VFA concentration (millimolar) decreased, and acetate-to-propionate ratio increased with higher dietary uNDF240, but peNDF content had no effect. Rumination time (min/d) was unaffected by diet, although eating time (min/d) was increased by both uNDF240 and peNDF content. An interaction between uNDF240 and peNDF resulted in lower dietary peNDF reducing eating and rumination time (min/kg of DMI) primarily for the HU diets. The HU diets increased the ruminal pool size of NDF and uNDF240 versus the LU diets, slowed NDF ruminal turnover rate, and increased apparent total-tract digestibility of potentially degradable NDF (NDF - uNDF240). Responses in DMI, ECM, chewing behavior, and ruminal characteristics reflected dietary particle size and fiber undegradability as measured by dietary uNDF240, peNDF, and peuNDF240 content.