The world No 10 has been upset by compatriot Taylor Fritz 7-6 (3) 7-6 (5) in a big serving battle.
"It feels great", Fritz said.
Leonardo Mayer saved three match points and fought from a set down to defeat Matteo Berrettini 5-7, 7-6 (5), 6-3, Jan-Lennard Struff thrashed Australian wildcard Rubin Statham 6-2, 6-1 while former world No. 3 David Ferrer received a guard of honour as he was forced to retire from his match against fellow Spaniard Pablo Carreno Busta.
The big American pulled the break back instantly with a forehand victor at the net after a well-constructed attack, with both players serving well in the remaining six games to set up a tie break where Taylor forged the lead with a mini-break in the very first point.
The 21-year-old will face a quarterfinal against the victor of Wednesday's match between Portugal's Joao Sousa and Cameron Norrie, an Auckland-raised player now based in England and playing under the Great Britain flag.
"We had a lot of close points".
The single break of serve was enough for the British wild card to claim the victory and set up a meeting with American Taylor Fritz in the last eight.
Fritz gained a mini break at 4-1, before two consecutive aces brought Isner back into contention at 4-4.
SPORTTaylor Fritz has one of the best records on the circuit in tiebreaks
This time Isner broke back for 4-4 but Fritz took a point against serve again at 6-4, sweeping a backhand victor across court as Isner advanced on the net.
Clarke looked to be fighting back in the second, breaking Sonego in the opening game, but could not hold on to the lead and was immediately broken back.
"I like to think that one of the strengths of my game is playing under pressure and playing the big moments well", Fritz said.
David tried to play but he chose to retire after he barely managed to move, receiving the special present in the post-match presentation after being one of the most successful players here, winning 32 matches in Auckland after the first appearance in 2003.
"So, I'm happy to start every year and today as well playing well under pressure and handling those big moments well".
Elsewhere, Germany's Philipp Kohlschreiber defeated Ugo Humbert of France 6-4, 6-4 to set up a quarter-final against Fognini.
Britain's Cameron Norrie secured his spot in the quarter-finals of the ASB Classic in Auckland with a straight-sets win over Joao Sousa.
After receiving medical attention, he returned to the court to play one more point before withdrawing.
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