The action, which runs from 4-7 June, is taking place at Kettering General, Leicester General, Leicester Royal Infirmary, Glenfield and Northampton General hospitals, which are run by University Hospitals of Leicester NHS Trust and University Hospitals of Northamptonshire NHS Group.
UNISON said healthcare assistants were only paid to provide personal care but are routinely being asked to undertake clinical tasks such as taking blood, performing electrocardiogram tests and inserting cannulas.
The union wants the salary band two workers to be upgraded to band three and for them to be given back pay compensation.
The three-day action was launched following the breakdown of mediation talks with Acas.
UNISON East Midlands regional secretary Chris Jenkinson said: ‘It's time for the chief executive to realise these NHS workers are in no mood to back down. He should make the healthcare assistants a reasonable offer that addresses years of historical pay inequality.'
Katarzyna, a healthcare assistant at Leicester Royal Infirmary, said: ‘The current back pay offer does not adequately compensate healthcare assistants for the years they worked without being paid properly. All we are asking for is to be fairly paid for the work we have done. No one wants to strike, but staff are determined to get what they deserve.'