Admin and clerical staff at Mid Yorkshire Hospitals Trust, which runs Pinderfields, Pontefract and Dewsbury hospitals, are taking action in an escalating dispute with managers.
Unions say changes to pay bandings to cut costs at the cash-strapped trust could leave staff up to £2,800 worse off.
The action follows a one-day strike on November 1, which was called after more than 70 staff were placed at risk of redundancy at Mid Yorkshire, which faces a £26m deficit at the end of the year.
More than 300 members of Unison and Unite were expected to take part in the strike.
Mid Yorkshire said contingency plans would be in place to ensure patient care was not affected.
The strike will end at midnight on Thursday.