Breastmilk iodine levels may vary temporally in response to recent changes in dietary iodine intake. We assessed the effect of and time to peak breastmilk iodine levels after potassium iodine ingestion, which has never been studied and is important toward interpretation of studies of breastmilk iodine measurements. Sixteen healthy lactating Boston-area women with no known thyroid disease were each given 600 μg oral potassium iodide (KI) (456 μg iodine) after an overnight fast. Iodine was measured in breastmilk and urine at baseline and hourly for 8 hours following iodine intake. All dietary iodine ingested during the study period was also measured. Mean age of mothers was 30.2±4.1 (SD) years. Median (interquartile range [IQR]) baseline breastmilk and urine iodine levels were 45.5 μg/L (IQR 34.5-169.0) and 67.5 μg/L (IQR 57.5-140.0), respectively. Following 600 μg KI administration, median increase in breastmilk iodine levels above baseline was 280.5 μg/L (IQR 71.5-338.0), and median peak breastmilk iodine concentration was 354 μg/L (IQR 315-495). Median time to peak breastmilk iodine levels following KI administration was 6 hours (IQR 5-7). Dietary iodine sources provided an additional 36-685 μg iodine intake during the 8-hour study. Following ingestion of 600 μg KI, there is a measurable rise in breastmilk iodine concentrations, with peak levels occurring at 6 hours. These findings strongly suggest that breastmilk iodine concentrations should be interpreted in relation to recent iodine intake.