Vaccination programs using live whole cell vaccines are presently being used to control outbreaks and to help protect flocks from field strains of Mycoplasma gallisepticum. The primary goal of the present study was to investigate the effects of a ts11-strain M. gallisepticum inoculation alone, and in conjunction with F-strain M. gallisepticum overlays at 22 or 45 weeks of age during lay. In this study, it was shown that a pre-lay ts11 vaccination had a broad range effect on the physiology of the commercial layer without affecting overall performance. The timing of an overlay vaccination using F-strain M. gallisepticum during lay also had varied effects on physiological parameters without affecting overall performance. This study indicates that using ts-11-strain M. gallisepticum in conjunction with F-strain M. gallisepticum does not negatively affect laying hen performance. Furthermore, this inoculation combination offers benefits above the use of those achieved through either inoculation alone