Cows managed for a 16-mo extended lactation have, at least, 8 estrus periods before being inseminated. The aims were to determine the effect of estrus number on milk yield and estrus expression, and to compare conception rates with the rates obtained during the previous 10 mo lactation. It was hypothesized that (1) milk yield will decrease during the day of estrus, and this loss will diminish with increasing estrus number; (2) postponing the insemination (AI) will increase the expression of estrus leading to a better detection and a higher conception rate. A total of 62 Holstein cows (30% 1st parity) were managed for 16 mo extended lactation. The 8 first estrus periods were determined by milk progesterone levels (<3 ng/L) and the day of estrus by visual observations (mucus consistency, blood in genital area, cow jump/stand/lay). Milk yield at ‘estrus day’ was compared with ‘normal day’ yield (average yield of d −4, −5 and −6 before ‘estrus day’). The percentage of cows expressing estrus was calculated using the behaviors mounting, being mounted or standing as criteria. Milk yield was analyzed using a linear mixed effects model while a chi-squared test was used for evaluating estrus expression and conception rate. The results showed a milk loss of 0.56 ± 0.19 kg per estrus day (P < 0.001) with no differences between the 8 estruses (P = 0.3). This negative estrus effect on milk yield is small compared with the well-known pregnancy effect. Moreover, 70% of the cows expressed the 8th estrus compared with 37% for the 1st estrus (P = 0.02) and 40% for the 2nd estrus (P = 0.06). However, the conception rates of the 1st and 2nd AI did not differ from those of the previous 10 mo lactation (respectively + 2.9%, P = 0.8 and + 3.4%, P = 0.7). To conclude, milk yield decreases similarly during each of the 8 estrus days studied whereas the proportion of cows expressing estrus nearly doubles from the 1st and 2nd to the 8th estrus.