Introduction: Breast milk is an excellent biofluid that ensures optimal growth, development, and strong immunity of the baby. Breast milk content may alter depending on duration and time of breastfeeding, the infant's age, and maternal health status. There are few studies in the literature investigating the effect of religious fasting on breast milk composition. Method: The study included 21 fasting and 27 nonfasting mothers who exclusively breastfed their babies in 2021 Ramadan month. The energy, carbohydrate, protein, and lipid levels of the collected breast milk samples and the macro- and micronutrient contents of the mothers' diets and the weight gain of the infants during the study were evaluated. Results: The mothers' ages, education levels, and weights at birth and at the time of sampling were similar. There was no significant difference between the energy, carbohydrate, protein, and lipid composition of breast milk in fasting and nonfasting mothers. Although daily energy, protein, carbohydrate, fiber, and vitamin intakes between two groups were similar, mean daily intake of lipid, sodium, chloride, iodine, and omega-3 fatty acids was determined significantly higher in study group. Also, there was no difference in the weight gain of babies during the month of Ramadan. Conclusion: Religious fasting of mothers does not affect the energy and macronutrient content of breast milk. In addition, fasting does not seem to affect the weight of mothers and babies.