Effects of dietary Original XPC (
XPC
) on 17 selected blood variables in commercial layer pullets challenged with the virulent, low-passage R strain of
Mycoplasma gallisepticum
(
R
low
MG
) were investigated. Hy-Line W-36 pullets sourced from
M. gallisepticum
–clean layer breeders were fed a basal diet with XPC (1.25 kg/metric ton) or without from hatch until 12 wk of age (
woa
). At 8 and 10 woa, half of the birds in each dietary treatment were challenged with R
low
MG. Blood samples were taken immediately before the initial R
low
MG challenge at 8 woa and again at 12 woa (4 wk after challenge). At 8 woa, blood pH was lower and glucose concentration was higher in the preassigned challenge treatment groups. At 12 woa, the concentration of oxygen dissolved in the blood was significantly lower in the R
low
MG-challenged group than the unchallenged group of birds regardless of dietary treatment. The R
low
MG challenge significantly increased blood carbon dioxide partial pressure, calcium, sodium, anion gap, osmolality, glucose, and corticosterone levels but significantly decreased blood oxygen partial pressure, oxyhemoglobin concentration, concentration of oxygen dissolved in the blood, chloride, and pH levels. Because blood pH and glucose concentration at 8 woa were examined before challenge, their baseline values were biased with respect to challenge treatment before treatment was applied. However, the lack of a significant main effect due to diet at 8 woa for blood pH and glucose concentration, along with the other 15 blood variables, indicate that the baseline data with respect to dietary treatment were unbiased, allowing for real dietary effects to be accurately assessed. In conclusion, layer pullets challenged with R
low
MG undergo a stress response associated with changes in various physiological blood variables, and a decrease in pH and increase in carbon dioxide partial pressure, in association with a lack of change in bicarbonate, indicates that the stress response caused by the R
low
MG challenge was associated with respiratory acidosis. Nevertheless, feeding XPC did not influence the effects of challenge treatment on these postchallenge physiological blood values.