Caledonian Sleeper staff set to strike at the end of the month
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The RMT union said working conditions on the overnight sleeper service between Inverness and London are causing "intolerable" stress.
It claimed poor staffing levels and insufficient training were among the issues operator Serco had failed to address.
Serco described the planned strike as "completely unnecessary" and said it was ready to hold further meetings with the union.
RMT members working on the sleeper service had previously voted by more than 10 to one to back industrial action.
The union has now instructed them not to book on shift from midday on Sunday, September 29 to 23.59 on Tuesday, October 1.
After the initial strike, RMT members have also been told not to work overtime, or on rest days, and not to "act up" in higher-grade roles.
RMT general secretary Mick Cash said: "The union is angry and frustrated that promises made to us to address the serious concerns of the Caledonian Sleeper staff at the end of July have not been honoured and that leaves us with no choice but to activate our industrial action mandate.
"RMT members have been put under intolerable personal stress as a result of the company's mismanagement of the sleeper service.
"They know it, they have promised to do something about it but they have failed to deliver."
He added: "The union remains available for genuine and serious talks."
The service has been hit by a series of problems since a revamped service launched in June
New "hotel-style" carriages are yet to come into service on the Highland route.
Caledonian Sleeper managing director Ryan Flaherty said the company was "disappointed and frustrated" by the threat of industrial action.
"We have met the RMT and company council on many occasions and the changes they have requested have either been completed or are under way," he said.
He said these included the recruitment of more staff at Edinburgh, Glasgow and London for a six-month period whilst the new Mark 5 carriages "settle in".
