ObjectiveTo compare the antibody response to influenza between health care workers (HCWs) who have received multiple vaccinations (high vaccination group) and those who have received fewer vaccinations (low vaccination group). DesignProspective serosurvey. SettingTertiary referral hospital. ParticipantsHealthcare workers. MethodsHealthcare workers were vaccinated with the 2015 southern hemisphere trivalent influenza vaccine. Influenza antibody titres were measured pre-vaccination, 21–28days post-vaccination and 6months post-vaccination. Antibody titres were measured using the haemagglutination inhibition assay. Levels of seropositivity and estimated geometric mean titres were calculated. ResultsOf the 202 HCWs enrolled, 182 completed the study (143 high vaccination and 39 low vaccination). Both vaccination groups demonstrated increases in post-vaccination geometric mean titres, with greater gains in the low vaccination group. Seropositivity remained high in both high and low vaccination groups post-vaccination. The highest fold rise was observed among HCWs in the low vaccination group against the H3N2 component of the vaccine. ConclusionsBoth high and low vaccination groups in our study demonstrated protective antibody titres post-vaccination. The findings from the current study are suggestive of decreased serological response among highly vaccinated HCWs. More studies with larger sample sizes and a greater number of people in the vaccine-naïve and once-vaccinated groups are required to confirm or refute these findings before making any policy changes.