Mycoplasma gallisepticum (MG) is a major and economically significant pathogen of avian species. When administered before lay, F-strain MG (FMG) can reduce egg production during lay, but the ts-11 strain of MG (ts11MG) does not exert this effect. Two trials were conducted to determine the effects of pre-lay vaccinations of ts11MG, MG-Bacterin (MGBac), or their combination, in conjunction with an FMG challenge overlay after peak production on the blood characteristics of commercial layers. In each trial, 160 mycoplasma-free Hy-Line W-36 layers were housed in negative-pressure biological isolation units (4 units per treatment, 10 birds per unit) from 9 through 52 wk of age (woa). The following vaccination treatments were administered at 10 woa: 1) Control (no vaccinations); 2) MGBac; 3) ts11MG; and 4) ts11MG and MGBac combination (ts11MG+MGBac). At 45 woa, half of the birds were challenged with a laboratory stock of high-passage FMG. Parameters measured in both trials were whole-blood hematocrit and serum concentrations of cholesterol (SCHOL), triglycerides, calcium, and total protein (STP). An age×treatment interaction (P=0.04) was observed for STP between 23 and 43 woa. The STP concentration in the ts11MG and ts11MG+MGBac groups was higher at 33 woa, but was lower at 43 woa, in comparison to the Control group. Also, at 38 woa, the STP of the ts11MG+MGBac group was higher than that of the MGBac group. Although use of the ts11MG vaccine alone or in combination with MGBac may influence circulating STP concentrations when administered before lay, it remains effective in protecting layers against the adverse effect of a post-peak challenge of FMG on egg production, as was observed in a previous companion study.