Infections of domestic and wild birds with low pathogenic avian influenza viruses (LPAIV) have been associated with protective immunity to subsequent infection. However, the degree and duration of immunity in wild birds from previous LPAIV infection-by the same or a different subtype-are poorly understood. Therefore, we inoculated H13N2 (A/Black-headed gull/Netherlands/7/2009) and H16N3 (A/Black-headed gull/Netherlands/26/2009) LPAIVs into black-headed gulls (Chroicocephalus ridibundus)-their natural host species-and measured the long-term immune response and protection against one or two re-infections over a period of more than one year. This is the typical interval between LPAIV epizootics in wild birds. Re-infection with the same virus resulted in progressively less virus excretion, with complete abrogation of virus excretion after two infections for H13 but not H16. However, re-infection with the other virus affected neither level nor duration of virus excretion. Virus excretion by immunologically na?ve birds did not differ in total level of excreted H13 or H16 virus between first- and second-year birds, but duration of H13 excretion was shorter for second-year birds. Furthermore, serum antibody levels did not correlate with protection against LPAIV infection. LPAIV-infected gulls showed no clinical signs of disease. These results imply that the epidemiological cycles of H13 and H16 in black-headed gulls are relatively independent from each other and depend mainly on infection of first-year birds.