Concentration of natural protective factors (NPFs) which have the ability to inhibit growth of mastitis-causing pathogens increase rapidly following the cessation of milking of dairy cows. One such NPF is lactoferrin, an iron-binding protein present in high concentrations in dry-cow secretions. Earlier studies have demonstrated that intermittent milking at the end of lactation increases levels of NPFs in milk and may decrease prevalence of intramammary infections at calving; however, most of these studies date back several decades and may not apply to current high-producing cows. The objective of this study was to assess whether an intermittent milking schedule prior to dry-off increases the concentration of lactoferrin in mammary secretions at the end of lactation and what other factors influence lactoferrin concentration at dry-off. One week prior to dry-off (pre-dry), cows were randomly assigned to an intermittent milking schedule or they continued to be milked twice daily. Duplicate quarter milk samples for microbiological culture were taken at pre-dry and at dry-off to determine infection status of quarters. Quarter somatic cell counts (SCC) were measured on the day of dry-off. Lactoferrin concentrations were quantified by ELISA. Intermittent milking, mean SCC for the last three months prior to dry-off, SCC at dry-off, lactoferrin concentration at pre-dry, quarter infection status at pre-dry and dry-off, days in milk at dry-off, breed, parity, cumulative milk yield for the final week of lactation and season were considered as potential explanatory variables. Their effect on lactoferrin concentration at dry-off was assessed using a mixed-effects linear regression model. Lactoferrin concentration increased significantly during the final week of lactation for cows on an intermittent milking schedule and was significantly associated with initial lactoferrin concentration and infection status at dry-off.