The seven-car maglev train -- short for "magnetic levitation" -- hit a top speed of 603 kilometres an hour, and managed nearly 11 seconds at over 600kph, operator Central Japan Railway said.
The new record came less than a week after the company recorded a top speed of 590 kph, breaking its own 2003 record of 581 kph.
The maglev hovers 10 centimetres (four inches) above the tracks and is propelled by electrically charged magnets.
"It gave me chills. I really want to ride on the train," an elderly woman told public broadcaster NHK as the carriage rocketed past her.
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"It's like I witnessed a new page in history."
An AFP reporter who previously rode on the super-speed train said the experience was like taking off in a plane, with the feeling of g-force gathering as the speedometer is pushed ever higher.
"The faster the train runs, the more stable it becomes -- I think the quality of the train ride has improved," Yasukazu Endo, who heads the maglev test centre southwest of Tokyo, told reporters today.
The service, which would run at a top speed of 500 kph, is expected to connect the two cities in only 40 minutes, less than half the present journey time in Japan's already speedy bullet trains.
By 2045, maglev trains are expected to link Tokyo and Osaka in just one hour and seven minutes, slashing the journey time in half.
However, construction costs for the dedicated lines are astronomical -- estimated at nearly $100 billion just for the stretch to Nagoya, with more than 80 percent of the route expected to go through costly tunnels.
He is due in the United States this weekend, where he will be touting the technology for a high-speed rail link between New York and Washington.