We compared the effect of order of administration of investigational alphavirus vaccines on neutralizing antibody response. Volunteers who received the inactivated eastern and western equine encephalitis (EEE and WEE) vaccines before live attenuated Venezuelan (VEE) vaccine had significantly lower rates of antibody response than those receiving VEE vaccine before EEE and WEE vaccines (66.7% vs. 80.6%; p=0.026). The odds of having a VEE antibody non-response among those initially receiving EEE and WEE vaccines, adjusted for gender, were significant (odds ratio [OR]=2.20; 95% CI=1.2-4.1 [p=0.0145]) as were the odds of non-response among females adjusted for group (OR=1.81; 95% CI=1.2-2.7 [p=0.0037]). Antibody interference and gender effect have major implications for vaccine strategy among those receiving multiple alphavirus vaccines and those developing next generation vaccines for these threats.