No V/Line trains will run on the Albury, Seymour and Shepparton lines all day on Thursday, as Rail Tram and Bus Union members take protected industrial action.
It comes after a two-hour delay and service cancellation on the Albury service on Wednesday, and a delay of nearly four hours on Monday.
V/Line chief executive James Pinder was in Seymour on Wednesday to speak to Thursday's planned disruptions.
"We're running a full coach replacement service tomorrow, so we can make sure we can get everyone where they want to be," he said.
"We know our customers prefer trains.
"We obviously regret the disruption this is causing.
"We're in talks today with the RTBU.
"We haven't been making a great deal of progress, if I'm honest, which is one of the reasons why I think the government has offered the assistance of an independent mediator.
"We welcome that offer, and hope that the union will take the government up on that offer soon so we can get to the end of this."
Mr Pinder said the negotiations with the RTBU were "not about money".
"There has been some discussions around modernising our agreement, and the way that we operate; this is nothing to do with paying people any less, cutting jobs out of the business, or removing allowances - this is all about modern working practices," he said.
"At this stage, we haven't even discussed in any great detail a pay offer as such, we're working within the confines of the Victorian public sector pay policy."
A Victorian government spokeswoman said safety was the government's top priority.
"The Government is pleased the RTBU has accepted the offer for an independent mediator to help bring about an end to this unnecessary industrial dispute," she said.
Delays experienced in recent days on the V/Line service have been unrelated to strike action.
Monday's 12.45pm Albury to Melbourne V/Line train was due to arrive at Southern Cross at 4.40pm but arrived at 8.18pm.
An ARTC spokesperson said a related signal fault was being investigated.
"We are currently working with V/Line to understand why this service was delayed more than others passing through Wallan and the matter is being investigated," he said.
"There are interim train rules in place for this location for rail operators to follow while repairs are made to a signal relay hut that was affected by an electrical fire."
Heather Howard was a passenger on the train, and wrote in a letter to The Border Mail that Monday's delay tied into an "ongoing problem".
"Many people were severely inconvenienced as they missed flights and connecting trains," she said.
"As this sort of transport is used predominantly by either the elderly or the young, it is imperative that a solution to these delays be done immediately."
Ovens Valley MP Tim McCurdy said it was unacceptable.
"This whole situation is an absolute disgrace, from the operation of V/Line, to the outdated carriages and these ongoing issues with the trains," he said.
After Monday's delays, the 6:35am Albury to Southern Cross service was delayed by two hours on Wednesday, and the 5.20pm service was replaced by coaches, both because of track congestion caused by a rail equipment fault in the Wallan area.
Tony, who did not wish to provide his last name, said his daughter was on Wednesday morning's service.
"She rings me at a quarter to one, very distressed," he said.
"She just said it was going that slow and stopping.
"I go to Melbourne regularly to see a specialist but I think I'll be flying.
"I am amazed to think how tourists from Japan or Germany would be thinking when they're riding on this archaic service."
IN OTHER NEWS:
Passengers are advised to allow up to an extra 60 minutes for their journey and to download the temporary replacement coach timetable from the V/Line website to plan their journey.
Freight trains managed by private operators and other services will still run during planned industrial action so the community is reminded to always obey warning signs at level crossings and stay safe around trains.
We would like to thank passengers for their understanding during the disruption.