Anti-p24 antibody response from primary FIV vaccination was higher than annual FIV re-vaccination. Microsphere immunoassay (MIA) detected increases in anti-p24 antibodies over time in both primary and annual vaccination groups (Primary: p < 0.0001; Annual: p = 0.012; blue and red bars below the box plots, respectively; mixed-effects analysis). Anti-p24 antibodies differed significantly over time between groups (interaction, p < 0.0001) and at individual time points following vaccination (p < 0.0001, where the black bars joining the box plots indicate the analysis of the comparative time points), exhibited by increased antibody levels in primary vaccinated cats compared to annual vaccinated cats (mixed-effects analysis). Samples from days 0, 14 *, and 28 † post-final vaccination were compared (Primary: days 0, 70 * and 84 †; Annual: days 0, 14 *, and 28 †). Negative fold (NF) = (sample MFI)/(2 × negative control MFI). OD = optical density. * = p < 0.05, **** = p < 0.0001.