Background and Objective: There is a question as to whether melatonin levels in breast milk are impacted by the cooling rate postpasteurization. Past research that has used in the Australian donor bank's breast milk Holder Pasteurization technique has reported varying findings regarding melatonin levels postpasteurization. Where breast milk was cooled slowly, a significant reduction in breast milk melatonin levels was observed. Conversely, where a rapid cooling method was used, there was no significant reduction in melatonin levels. The aim of this study was to investigate whether the cooling process between the different pasteurization techniques impact on melatonin levels in breast milk postpasteurization. Materials and Methods: Twenty-seven nighttime breast milk samples were collected, with each sample divided into three; one remained unpasteurized, one was pasteurized and rapidly cooled to 4°C, and the other was pasteurized and cooled slowly to 4°C. Results: Melatonin levels were significantly reduced in both the rapidly cooled and slow cooled breast milk samples when compared to their unpasteurized counterpart (p<0.001). There was no significant difference in melatonin levels between the two cooling methods (p=0.91). Conclusion: This study showed that both the rapid and cooling pasteurization processes had a similar reduction in melatonin levels in breast milk. However, even after pasteurization melatonin was still present. Therefore, it is recommended that donor banks still take into consideration circadian timing hormones such as melatonin and the time of day breast milk is expressed.