Ultimately, it was Anderson who prevailed, taking the match 7-6, 6-7, 6-7, 6-4, 26-24.
Isner won the longest match at Wimbledon - or any other tournament - when he beat Nicolas Mahut 70-68 in the fifth set after 11 hours, 5 minutes in 2010.
Eighth-seeded Anderson's prize for his bittersweet victory over his former U.S. college friend and rival is a date on Sunday with either world No.1 Rafael Nadal or fellow grand slam giant Novak Djokovic.
It was the longest semi-final ever played at Wimbledon, surpassing the four hours 44 minutes it took Djokovic to beat Juan Martin del Potro in 2013.
With a spot in the Wimbledon final on the line, the two power players beat each other to a standstill in a match that stretched nearly seven hours.
Maybe the most excruciating part of all is that Anderson now has to turn around and get ready for the championship on Sunday, while Isner has to face the harsh reality that this preposterous marathon match was all for naught.
"John is a great guy and I really feel for him".
"I think going into the finals, there was maybe a bit too much relief that, "Hey, I'm in the finals" and didn't give myself as much of a chance to win that match from a mental side".
Anderson had landed on his back giving a return from the baseline, losing his racquet in the process.
He is yet to be broken in this competition and though this is his first Wimbledon semi-final he will be a force.
On Friday, Isner will play in his first Grand Slam semifinal against South African Kevin Anderson - who yesterday knocked World No. 1 Roger Federer out of the tournament in a stunning upset.
It is common to hear the call of 15-all in tennis, but, in this instance, that was the score in games at one stage in the fifth set as both players continued to resolutely hold serve.
The 33-year-old American´s composure wasn´t helped by a delay caused by a ball boy slipping and falling.
The Canadian and partner Yifan Xu of China, seeded sixth, lost 6-3, 4-6, 7-5 to No. 12 seeds Nicole Melichar of the US, and Kveta Peschke of the Czech Republic in the Wimbledon semifinals on Friday.
He fashioned three break points from the next rally and took the second, the first break in 49 games. Isner in particularly started to appear tired at the set wore on. It was a drama filled set that went back-and-forth, similar to the third set.
But this time he dug deep to break again in the ninth game on his way to levelling the semi-final at two sets apiece.
Anderson notably broke Isner in the eighth game of the third set to lead 5-3, only to play an ill-advised forehand in the next game, which allowed his opponent to roar back from 15-30 down and break right back.