The match proved far closer than Thiem's attempt to unseat Nadal in the French Open final earlier in 2018, when the Spaniard won 6-4 6-3 6-2.
In an interview after his titanic battle, the 32-year-old Nadal gave credit to his opponent saying the match was very demanding in all aspects after almost five hours on court.
American star Isner added: 'It's tough conditions out there, no doubt.
"To reach the semifinal again here in NY in my favourite tournament is very special to me", said Del Potro after his eighth win over Isner in 12 meetings.
"I'm sad for him because when this moment arrived, he did all the things well to win the match".
"He's young, he has plenty of time to win big tournaments", he said on court.
"He has a great attitude and is a great fighter".
Under the lights of Arthur Ashe Stadium, Nadal and Thiem delivered the NY crowd another memorable marathon, this time for a spot in the US Open semifinals.
Over 500 flights cancelled as Typhoon Jebi bears down on Japan
A auto lies upside-down along a road after being blown by typhoon Jebi's strong winds in Osaka on September 4, 2018. The storm also did serious damage to Kansai International Airport, one of Asia's most important airports.
"Thank you for the all the support", he said, addressing the Arthur Ashe crowd. "I'm happy that I did this for the first time, even if it went the wrong way".
This was Nadal's 247th win at Grand Slams and the 29th semi-final, moving ahead of Ivan Lendl and standing just two behind Jimmy Connors in the third place, with a nice chance of catching him in 2019. The momentum was with Nadal but Thiem regrouped in the third set and began to again blast winners into the corners.
In the fifth, Nadal held three break points at 5-all, love-40, but Thiem took the next five points told serve.
"I don't think he is sorry", the Austrian said with a laugh.
Next up for Serena is a quarterfinal meeting with eighth seeded Czech Karolina Pliskova who advanced with a 6-4, 6-4 win over 18th seed Ashleigh Barty of Australia.
Perhaps Thiem, after a change of socks and shoes, looked the fresher going into the decider but he needed to save two break points at 2-2, one with an ace, to maintain parity.
Nadal has the best part of two days to recover before a semi-final against Juan Martin Del Potro, while the world No 9 left a stadium largely drained of spectators with nothing more than a reminder of his opponent's incredible fighting qualities.
World #1 Rafael Nadal will look to continue his US Open title-defence when he takes on ninth seed Dominic Thiem, who is competing in his first Grand Slam quarterfinal away from the clay and a fourth overall. "Me, too. I fought until the end. I hope that we have many more in the future with a different end". "Forever I'm going to remember this match, for sure", said Thiem after his first ever hard court match against Nadal.
Nadal ran into an inspired opponent for the second match in a row and was put through his paces by Nikoloz Basilashvili before eventually coming through 6-3, 6-3, 6-7, 6-4.