Background: Milk ejection characteristics remain consistent throughout 12 months of lactation in women who expressed breastmilk with an electric breast pump. In addition these characteristics appear to remain constant when women are breastfeeding or pumping suggesting that milk ejection is a robust physiological response. It is not known whether the stimulation of an infant at the breast in the early post partum period influences milk ejection patterns or whether this is a programmed event. However, as more data become available on the mechanisms involved in infant feeding, pumping patterns mimicking the infant more closely may provide enhanced results. The objective of this study was to compare milk ejection characteristics obtained when using a novel infant-derived pumping pattern with an established 2-phase pattern. Methods: A convenience sample of ten lactating mothers, 1 to 40 weeks of lactation with normal milk production were recruited in 2015. Each participated in two pumping sessions in which either a 2-phase pattern or infant-derived pattern were randomly assigned. Milk volume and milk ejection characteristics were recorded and the percentage of available milk removed (PAMR) was calculated. Statistical analysis used linear mixed effects modeling to determine any differences between breasts and pump patterns with the consideration of individual variability as a random effect. Results: The number of milk ejections and milk ejection characteristics did not differ between patterns. Milk volumes removed were 53.6 ± 28.5 ml (PAMR 58.2 ± 28.4) for the 2-phase pattern and and 54.2 ± 26.3 ml (PAMR 52.2 ± 22.3) for the infant derived pattern. Peak milk flow rates were positively associated with the available milk (p = 0.0003) and PAMR (p = 0.0001), as was the volume of milk removed during each milk ejection (p = 0.001 and p = 0.0001). Conclusion: An experimental pumping pattern designed to resemble infant sucking characteristics did not alter milk ejection characteristics or milk removal parameters compared with an established 2-phase pattern. Theses findings provide further evidence that milk ejection is a robust physiological response.