Milk ejection is essential for effective milk removal during breastfeeding and pumping, and for continued milk synthesis. Many women are unable to accurately sense milk ejection to determine whether their infant is receiving milk or, when pumping, to switch the pump to a more effective expression pattern. To determine if changes in bioimpedance parameters are associated with milk ejection in the lactating breast during pumping. 30 lactating women participated in 2 pumping sessions within 2 weeks of each other. During pumping the breasts were monitored with bioimpedance spectroscopy (on either the pumped or the non- pumped breast), and milk flow rate and volume were measured simultaneously. All mothers completed 24-h milk productions. Linear mixed effects models were used to determine associations between milk flow rate and bioimpedance changes. Changes in bioimpedance parameters were greater at the first milk ejection when measured on the pumped breast (median (IQR): R zero: −7 (−17, −4,) % (n = 30); R infinity: −8 (−20, −2) % (n = 29); membrane capacitance: −24 (−59, −7) % (n = 27). Changes in bioimpedance detected in the non-pumped breast were lower at the first milk ejection, R zero: −3 (−8, −2) % (n = 25); R infinity: −5 (−8, −2) % (n = 23); membrane capacitance: −9 (−17, 15) % (n = 24). Smaller less consistent decreases in the bioimpedance characteristics were detected at the second milk ejection in both breasts. Bioimpedance parameters showed a consistent decrease associated with the first milk ejection when electrodes were placed on the pumped breast. Smaller decreases were observed when the non-pumped breast was monitored for the first and second milk ejection. There was wide variation in the magnitude of changes observed, and hence further development of the methodology is needed to ensure reliability.