Background Maternal avoidance of egg intake has been recommended to treat egg allergy in breastfed infants. Objectives To determine if the concentration of ovalbumin (OVA) in human milk is directly related to the quantity and form of egg consumed by breastfeeding mothers. Methods Randomized, blinded, cross-over, intervention trial. Breastfeeding women (n=41) attended four clinic days between 11 and 14 weeks of lactation and on each day were randomly allocated to receive a test breakfast, identical except for the egg content (no egg, one raw egg, half a cooked egg or one cooked egg). Breast milk samples were collected at two hourly intervals for 8 h and their OVA concentration measured by ELISA. Results There was a direct, dose-response between the amount of cooked egg ingested and the peak OVA concentration (no egg 0.05 ng/mL [95% confidence interval (CI), 0.01-0.11], half a cooked egg 2.24 ng/mL [95% CI, 0.57-3.91], one cooked egg 3.16 ng/mL [95% CI, 1.41-4.91], n=41, P<0.05) as well as the total OVA excretion (no egg 0.18 ng/mL/h [95% CI, 0.04-0.39], half a cooked egg 4.93 ng/mL/h [95% CI, 1.40-8.46], one cooked egg 9.14 ng/mL/h [95% CI, 4.25-14.03], n=41, P<0.05). The peak concentration and total OVA excretion in response to one raw egg did not differ from ingesting half a cooked egg. There was no detectable OVA in the breast milk of 24% (10/41) women up to 8 h after any egg challenge. Conclusion OVA was detected in the breast milk of lactating women up to 8 h after a controlled intake of egg. A dose-response correlation was indicated. As excretion of OVA in human milk appears to be a normal phenomenon, further studies need to determine the threshold of OVA excretion that leads to symptoms in egg-allergic breastfed infants.