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The objective of this observational study was to evaluate the association of estrous expression within 50 days in milk (DIM) using a neck-mounted automated activity monitor (AAM; Heatime Pro; SCR Engineers Ltd., Netanya, Israel) and reproductive performance in lactating Holstein cows. A total of 3,334 lactating cows (1,017 primiparous cows and 2,317 multiparous cows) from 7 commercial dairy farms were included in the statistical analyses. Cows were classified according to the number of estrus events from d 7 until d 50 into 4 categories: 1) no estrus event (ANESTRUS); 2) one estrus event (ESTRUS1); 3) at least two estrus events (ESTRUS2+). Generalized linear mixed models were used to analyze continuous or categorical data. Shared frailty models were used for time to event data. Overall, 41.9% (1,396/3,334) of cows had no estrus event detected by an AAM system from d 7 until d 50. Estrous expression within 50 d affected estrous duration (P = 0.001), estrous intensity (P = 0.001) and P/AI (P = 0.001) at first AI. Estrous expression within 50 d affected time to first AI (P = 0.001) and time to pregnancy (P = 0.001). Median DIM to pregnancy were 136, 118, and 103 for ANESTRUS cows, ESTRUS1 and ESTRUS2+, respectively. In conclusion, cows with no estrous expression from 7 to 50 DIM had reduced estrous expression at first AI and inferior reproductive performance compared with cows that displayed estrus activity. Future studies should address risk factors for ANESTRUS and evaluate intervention strategies in these cows to improve their reproductive performance.