With world number one Rafael Nadal waiting for the victor, Tuesday's fourth set, played in much cooler conditions than the first three the day before, turned into a mini-epic before Del Potro eventually prevailed 7-6 (1) 7-6 (5) 5-7 7-6 (5).
5-7 win over Frenchman Gilles Simon after fading light interrupted their last-16 clash the previous evening with the fifth seed up two sets to one. A former champion saved more strength for the final set, breaking del Potro in game five and saving two break points in the following game to move 4-2 up and cement his break.
When they came back out on Tuesday, Del Potro completed a 7-6 (7-1) 7-6 (7-5) 5-7 7-6 (7-5) victory on Court Two, but only after sealing the win with his fifth match point. He must get past three-time champion Novak Djokovic in the semi-finals on Friday for another shot at the title.
The world number one, playing in his first All England Club quarter-final since finishing runner-up to Djokovic in 2011, then wasted four set points in the second.
As he served out to reach his sixth semi-final, his old sparring partner, Andy Murray, newly installed in the BBC commentary box, declared the quality of tennis in the fifth set "the best I've seen in the tournament so far".
There was, however, one consolation for Del Potro who came off second best despite belting down 33 aces and producing 77 staggering winners, 10 more than Nadal.More news: Kylie Jenner Covers Forbes As The Next Youngest Self-Made Billionaire
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The dogfight Nadal feared and Del Potro knew was his only route to the semi-finals had begun in earnest.
"In the end the court was a little bit slippery because the sun goes out and it was humid on the grass", Del Potro added.
"Rafa is a fighter", del Potro said.
Nadal broke for 3-2 but had to save break points in the sixth and eighth games before securing a memorable victory. The hard-serving American beat Milos Raonic of Canada 6-7 (5), 7-6 (7), 6-4, 6-3 to set up a meeting with Kevin Anderson of South Africa in Wimbledon's last four.
Isner has yet to be broken at this year's tournament and saved the only break point he faced on Wednesday.