Geoff Marshall, 41, from Ealing, west London, and Anthony Smith, 28, completed the record in 16 hours, 20 minutes and 27 seconds.
It is the second time Mr Marshall has held the record, after a total of 25 attempts.
The freelance video producer said it took “more skill than people think”.
“You have to get off one line and run to the other,” Mr Marshall said.
“We run from Cockfosters to High Barnet so it’s a combo of athleticism and the ability to decode a Tube timetable.
“People tar you with a trainspotter brush, but I’ve never written down a train number in my life.”
The pair’s attempt was completed on 16 August, but it took Guinness World Records more than a month to verify their record.