When asked how he would recover from such an epic and play in the Wimbledon finals on Sunday afternoon, the South African said: "It's tough, I really don't know".
They have met on 51 previous occasions, but not at this stage of a Slam since the 2014 French Open semi-finals.
Twitter was abuzz with jokes that the Anderson-Isner match could well stretch beyond the 70-68 final-set scoreline that Isner won with over Mahut eight years ago.
At his 41st major tournament, Isner, 33, was playing his first Grand Slam semifinal and was trying to become the first American man since 2009 to reach a major final.
The pair will face off for a record 52nd time in their careers in the other men's semifinal on Friday evening.More news: Stormy Daniels Arrested In Ohio Strip Club, Michael Avenatti Says
The show being put on by Novak Djokovic and Rafael Nadal was so good it could have been an instant classic had they been able to finish their semi-final before the All England Club's official 11pm curfew.
Anderson and Isner go way back, to their days when they played in college, Isner at Georgia, Anderson at IL.
The players exchanged breaks again at the beginning of the fourth set, before Anderson grabbed a crucial break at 4-4 and served out.
They slugged it out on Centre Court for six hours and 35 minutes before the eighth seed came through 7-6 (8-6) 6-7 (5-7) 6-7 (9-11) 6-4 26-24.
LONDON-John Isner already had won the longest match in Wimbledon - and tennis - history.More news: United States tries to bully World Health Organization into dropping breast-feeding resolution
In a match between two of the game's biggest servers, Anderson started the match with a double-fault.
But Isner, after missing his first serve, sent up a bold swat of a second serve of 129mph to level at deuce.
This set was a departure with Anderson, thanks to some wonderful returns of serve, breaking to go up 3-2 only to find Isner breaking back instantly. With the set tied 7-7, Anderson had the chance to break Isner but the American fought him back to take an 8-7 lead, and so it continued as neither player seemed willing to blink.
He is yet to be broken in this competition and though this is his first Wimbledon semi-final he will be a force. "At the end, you feel like this is a draw".
Anderson finished with 49 aces and 118 winners; Isner had 53 aces and 129 winners.More news: Trump, after questioning May's Brexit plan, arrives in 'hot spot' UK
At one point in the fifth set, a spectator shouted, "Come on, guys!"