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The objective of this study was to determine the level of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in human milk in the first 2 weeks of breast-feeding and compare the effects of Holder pasteurization (HoP, 62.5 °C, 30 minutes) and microwave pasteurization (MP) at constant temperature (62.5 °C) on the concentraion of both neurotrophic factors (NFs). Concentration of NFs in human milk was determined using a commercially available ELISA assay. The average concentration of BDNF and GDNF in milk was 11 ± 6 ng/mL and 336 ± 238 pg/mL, respectively. There was a positive correlation between the concentrations of BDNF and GDNF in human milk and day of lactation (r= 0.441, P< 0.05 and r= 0.482, P< 0.05, respectively). In addition, there was a significant correlation between the levels of BDNF and GDNF (r= 0.366, P< 0.05). HoP and MP for 10 minutes affected both NF levels similarly, causing degradation of BDNF by about 24% and 17%, and GDNF by 47% and 45%, respectively. Use of MP for 5 minutes resulted in preservation of nearly 91% BDNF and 79% GDNF in human milk. In the pasteurization processes carried out, results showed that GDNF is more susceptible to degradation under the influence of high temperature. To the authors’ best knowledge, this is the first study evaluating the effects of HoP and MP at constant temperature on the concentration of NFs in human milk. It was found that the MP for 5 minutes is the optimal method.