Breastfeeding (BF) neonates generally lose weight after birth. Diurnal and nocturnal factors as well as season may affect BF. The aim of this study was to determine the effect of birth time and seasonal variations on weight loss in BF new-borns. In this retrospective study, two sets of groups were evaluated. Birth time groups (night time: 7 pm-7 am and day time: 7 am-7 pm) and seasonal variations groups (summer, autumn, winter and spring). Weight loss of more than 5%, 7%, 10% at <24 h, <48 h and <72 h of birth respectively was considered as a significant weight loss. 2044 newborns were analyzed. Preterm, exclusive formula fed new-borns and NICU admissions were excluded. In the birth time group, babies born during the night time had significantly lost >5% of birth weight at <24 h (p<0.01) and >7% weight at <48 h of life (p<0.02). Weight lost >10% at <72 h was similar in both birth time cohorts. C section, prolong rupture of membrane and maternal pre-delivery hospital stay for >12 h were significant contributing factors. Seasonal variations were not associated with weight loss in new-borns. Conclusion: BF babies born at night time lose significant weight during the first two days of life. Seasonal variations did not affect weight loss.