After the second set they both left the court for what turned out to be a six-minute bathroom break, with the usually mild-mannered Argentinian seemingly unhappy about the rules that were followed, remonstrating with officials as he came back out.
Despite that, the 29-year-old was never broken in the 3 hour 31 minute match where Isner unleashed 26 aces but was undone by 52 unforced errors compared to Del Potro's 14.
"I'm so happy to reach another semifinals in my favourite tournament", the 2009 champion said in his on-court interview.
"To play John in these kind of matches, it's like an epic". We both were exhausted in the end, we both fought until the end and I think I survived with my serve.
The players took a 10-minute break at the end of the third set, and Del Potro said: "I lay down on the table and I didn't want to come back again because with this heat it's impossible to play tennis".
The Argentine had the sole break chance in the opening set but the third seed could not convert, while Isner had a mini-break at 3-3 in the tiebreak and turned that into a 1-0 lead.
As the intense heat at FLushing Meadows continued Del Potro and Isner exchanged big serves in what was at times a fairly attritional affair.
"It was too hot for tennis".
The No 3 seed then held on to an early break gained in the fourth, despite needing courtside treatment.
He comfortably levelled the contest, giving up just three points on his own serve in the set.
It was enough to see 2009 champion Del Potro through to the last four in NY for a third time, and second in succession.
Isner, meanwhile, will return to Dallas as quickly as he can where his wife is due to give birth to their first child in two weeks' time.
Nadal beat Del Potro in the semi-finals last year at Flushing Meadows, and again this year in the semi-finals of the French Open.
The popular Argentinian will now attempt to take a step he has not managed since winning his only grand slam title here nine years ago and reach another final.
Del Potro now has two days off before the semi-finals but will be keeping to himself, saying: "If I met them it could be unsafe for me".
Isner was trying to become the first American man to reach the US Open semifinals since Andy Roddick in 2006.
Wednesday's quarter-finals will see two-time champion Novak Djokovic face Australia's John Millman, the world number 55 who shocked Roger Federer in the last 16.