Wimbledon champion Petra Kvitova became the fifth top 10 seed to exit the US Open in the first week on Saturday when she was knocked out by Serbian world number 145 Aleksandra Krunic.
Third seeded Kvitova lost the third round tie 4-6, 4-6 to the pint-sized 21-year-old from Belgrade, who came through qualifying and had never won a match at a major before this year's event.
Kvitova joins second seed Simona Halep, fourth-seeded Agnieszka Radwanska, Angelique Kerber, the sixth seed, and eighth-seeded Ana Ivanovic in failing to make the fourth round.
The 24-year-old Kvitova dropped serve five times and committed 34 unforced errors against the 5ft 4in (1.63m) Krunic who divides her time between the pro tour and studying for a university economics degree.
"It was an honour to play on the same court as Petra, she's a great champion. I watched both her wins at Wimbledon," said Krunic.
"I wanted to be myself on court, play as long as possible. I didn't expect to win. I hoped to win a set."
Krunic said she tried not to think about nerves even as a 4-2 lead in the second set was swallowed up by Kvitova who pulled level at 4-4.
But the slender Serbian broke for 5-4 and claimed her famous win on a second match point when another Kvitova forehand sailed wide.
"On match point I thought 'Don't serve a double fault.' I just wanted to keep the ball in until she missed," added Krunic who will next face either former world number one Victoria Azarenka of Belarus or Russian Elena Vesnina for a quarter-final berth.
Later Saturday, top seeds Novak Djokovic and Serena Williams target last-16 spots.
Djokovic, the 2011 champion who has made the last four finals, takes a 7-1 career lead over Sam Querrey, just one of two Americans left in the draw, into their third round clash.
The Wimbledon champion is looking to make the fourth round at a major for the 22nd consecutive time, a run stretching back to a third round exit at the 2008 French Open.
Williams, meanwhile, faces her third successive American opponent in the shape of Uzbekistan-born left-hander Varvara Lepchenko.
The top seed is looking to claim her 75th match win at the US Open.
Australian Open champion Stan Wawrinka, the third seed, faces Slovenia's Blaz Kavcic, the world number 92, who has never defeated a top-10 opponent in 10 attempts.
Andy Murray, the 2012 champion, aims for a seventh appearance in the fourth round when he takes on Russia's Andrey Kuznetsov, who knocked world number five David Ferrer out of Wimbledon this year.
Canadian fifth seed Milos Raonic faces Victor Estrella Burgos, the 34-year-old tournament debutant who is the first man from the Dominican Republic to play at a major.