Project chiefs said the trial would help make hydrogen trains north of the Border “a reality in the not too distant future”.
It follows the first hydrogen train entering passenger service last month on a line west of Hamburg in Germany.
Hydrogen is being considered for a new ScotRail fleet on Highland and other long-distance rural lines where electrification of the routes is not seen as cost-effective.
It could come as part of all the operator’s diesel trains being scrapped by 2035 in the most radical shift since steam engines were replaced with electric trains in Glasgow in 1960.
The zero-emission power source is already in use in double decker buses in Aberdeen and the city’s car-sharing club vehicles, while a fleet of hydrogen-powered bin lorries are being built for Glasgow City Council.
The Scottish Hydrogen Train Project is using a 40-year-old electric train that used to run in Glasgow, which was put on static display at Bo’ness during the COP26 climate change conference in Glasgow last November.
It is being powered by green hydrogen produced at Bo’ness by extracting the fuel source from water using electricity.
The project is being spearheaded by the University of St Andrews along with Transport Scotland, Scottish Enterprise and Ballard Motive Solutions, formerly Arcola Energy, which said no main line testing was planned “at this stage”.
Footage of the train running under hydrogen power has been posted on YouTube by David Horne, engineering inspector of the railway, which is run the Scottish Railway Preservation Society and offers steam and diesel train trips on Tuesdays, Saturdays and Sundays until the end of October.
A University of St Andrews spokesperson told Scotland on Sunday: “Planned testing of the innovative hydrogen-power train on the Bo’ness heritage line has been taking place throughout August and will continue in to the autumn.
"Testing is a demonstration of clean energy and is the next step in realising how we can make a green energy transition.
"The project, led by the university, is on track to deliver the critical understanding and knowledge to make hydrogen power rail a reality in the not too distant future.
"Tests underway use green hydrogen made on site and funded by Angel Trains in support of the project.
“The project entails the conversion and re-use of a 40-year-old three car class 314 train to a class 614 hydrogen fuel cell electric powertrain.”
However, the scheme has been criticised by Neil Robertson, the Scottish-born chief executive of the National Skills Academy for Rail, as “slightly ideologically driven” since similar projects are already underway south of the Border.
Train leasing firm Porterbrook showed off its Hydroflex hydrogen train during COP26 but said it had not been powered by hydrogen on demonstration trips around Glasgow so the interior of the train’s “ground-breaking” hydrogen tanks could be viewed.