Second seed Nadal lost a mere seven points on serve in the first two sets, struck 37 winners and barely put a foot wrong, comfortably sealing a last-eight meeting with Juan Martin Del Potro or Gilles Simon.
In a match suspended from Monday night due to darkness with the Argentine star two sets to one ahead, Del Potro sealed victory on a fifth match point after nearly four and a half hours of action on Court Two.
The bar was set absurdly high by the time Rafael Nadal walked onto Centre Court on Monday but the Spaniard responded with a sensational 6-3 6-3 6-4 defeat of Jiri Vesely to reach the Wimbledon quarter-finals.
Del Potro threw everything he could at Nadal in a pulsating match and even conjured the shot of the tournament as he acrobatically dived to his right to hit a fizzing forehand volley that ended a spellbinding 23-shot rally. In the next game, Nadal staved off two break points, the first when he used his never-give-up-on-a-ball defence to stretch a point and del Potro pushed a forehand way long.
Next up for the Spaniard is a blockbuster last four showdowns with Serb Novak Djokovic.
But Nadal was first to break in the deciding set and he built a 5-3 advantage with a looped forehand victor before confirming his final four place with a volley after four hours and 48 minutes. The set stayed on serve until Del Potro secured the first break point for a 4-3 lead.
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Nadal caught Del Potro napping in the fifth game of the fourth set, breaking him to love to lead 3-2.
The dogfight Nadal feared and Del Potro knew was his only route to the semi-finals had begun in earnest.
"Rafa is a fighter", del Potro said.
"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.
Nadal slipped mid-court twice in the fifth game, but stayed upright to break with a cracking angled backhand. He went 4-3 in front, before closing out the set to increase the pressure on Nadal.
Isner's 41 attempts to make a semifinal at a major is the second most in the 50-year professional era, behind Sam Querrey, who made the last four at Wimbledon last year with his 42nd try.