Inconsistent information exists regarding indicators of secretory activation in mothers delivering very low birth weight infants. To compare time to the onset of secretory activation using three separate indicators. A secondary aim examined the association between indicators of secretory activation and milk production. Indicators of secretory activation included maternal perception, volume attainment (production of ≥ 20 mL in two consecutive expression sessions) and biomarkers (sodium and lactose) obtained at volume attainment. Milk production was measured on Days 1-7 and then weekly for 6 weeks. In 69 mothers of infants born ≤ 32 weeks' gestation and < 1500 g, we found no correlation in time to secretory activation between indicators. Earlier volume attainment was associated with increased milk production on Days 1-7, 14, 21, and 28 (all p < .007). Participants who exhibited both normal lactose and sodium levels produced more milk on Days 28 and 42 (p = .028 and .011), those with only normal lactose levels produced more on Day 42 (p = .026) and those with only normal sodium levels on Day 28 (p = .036). Earlier secretory activation by volume attainment was associated with increased expression frequency during Days 2-5 (all p < .014) and participants with normal biomarkers expressed more frequently during Days 2-5 (all p < .020). Mothers of very low birth weight infants are at risk for delayed secretory activation, which may decrease their milk production. Frequent expression during the first 5 days postpartum may promote earlier secretory activation. Valid methods of determining secretory activation are necessary to develop interventions promoting earlier secretory activation.