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Early life environmental events mediated by maternal care have a lasting effect on adult phenotype through epigenetic modifications. Using brief maternal deprivation as early life stress model, we previously revealed immediate and long-term adverse effects on physical development, social behavior, and learning, as well as on the level of histone acetylation in the neonatal brain. However, behavioral and hormonal factors which trigger these changes have not yet been identified. As such, we examined two early life stress markers: ultrasonic vocalizations (USV) rate on P6 and the blood corticosterone level on P7, and also changes in maternal behavior on P6 after 45-min/day repeated maternal deprivation on postnatal days P3-P6. We have shown, for the first time, that 45-min maternal deprivation for 4 consecutive days causes stress response in neonates, which is accompanied by changes in maternal behavior. Importantly, increase in pups’ blood corticosterone level and USV rate showed no recovery to the baseline level 30 min after the reunion. These results suggest that behavioral and hormonal responses described above might be the early life predictors of long-term adverse effects of maternal deprivation. It is important to take into account these findings when performing different experimental manipulations of pups, if these procedures require brief pup removal from the mother in the early postnatal period. Supported by RFBR Grants 14-04-01768 and 16-34-00253