The importance of an efficacious, long acting foot-and-mouth disease (FMD) vaccine is critical to control FMD. The human replication deficient adenovirus 5 (Ad5) empty capsid FMD virus (FMDV) platform (AdtFMD), is a promising new vaccine technology. Herein we describe three studies assessing the proportion of animals protected from clinical vesicular disease (foot lesions) following live-FMDV challenge by intradermolingual inoculation at 6 or 9 months following a single vaccination, and the potential effect of vaccination route (transdermal, intramuscular, subcutaneous) on clinical outcome. Results demonstrate that a single dose AdtA24 vaccination in cattle induced protection against clinical FMD at 6 months (100% transdermal, 80% intramuscular, and 60% subcutaneous) that waned by 9 months post-vaccination (33% transdermal and 20% intramuscular). Post-vaccination serum from immunized cattle (all studies) generally contained FMDV specific neutralizing antibodies by day 14. Overall, when specific Ig isotype responses were assessed, the number of IgG1 anti-FMDV antibody secreting cells detected in vaccinated steers compared to placebo-immunized controls was statistically significant (p=0.007). The decay in protective immunity over time may be a function of FMDV-specific antibody half-life due to loss of antibody secreting cells in circulation by four to five weeks post vaccination. These data reveal important performance characteristics of needle-free vaccination with a recombinant vectored subunit vaccine for FMDV.