No.1 seed Karolina Pliskova has suffered a shock 6-1 7-6 (7-2) loss to Frenchwoman Caroline Garcia to crash out of the US Open.
Pliskova, a former world No.1, had no answer to the power of Garcia who fired 30 winners to the Czech's 13 on Wednesday.
"This (seeding) has nothing to do with my loss today," Pliskova told reporters.
"I think there are just some girls who are playing good tennis ... Garcia is one of them."
In a day of upsets, seeds Elena Rybakina (11), Marketa Vondrousova (12) and Alison Riske (13) all bowed out.
Shelby Rogers defeated Rybakina 7-5 6-1 while Ann Li beat Riske 6-0 6-3.
French Open finalist Vondrousova was beaten by Belarusian Aliaksandra Sasnovich 6-1 6-2.
Naomi Osaka maintained her winning streak with a statement 6-1 6-2 victory over Italian big-hitter Camila Giorgi.
Osaka, who played with her left hamstring strapped, dropped a mere five points on serve.
Two-time Wimbledon champion and Czech sixth-seed Petra Kvitova overcame a bout of nerves and a rocky start to defeat Kateryna Kozlova 7-6 (7-3) 6-2 in her second-round clash.
Former champion Angelique Kerber came through a fierce all-German tussle against Anna-Lena Friedsam 6-3 7-6 (8-6) to book her place in the third round.
France's Kristina Mladenovic complained of living a "nightmare" due to the COVID-19 health protocols in New York after suffering a spectacular second-round meltdown to Russian Varvava Gracheva.
Mladenovic looked set for a breezy win while serving at 6-1 5-1 but things went downhill for her abruptly and she ended up losing 1-6 7-6 (7-2) 6-0.
"It's absolutely abominable how they are treating us, but I don't want that to be an excuse for my defeat," she said.
"It's not the USTA's fault that I didn't convert four match points, don't get me wrong.
"At 5-2, I just collapsed. I have nothing more to say, I'm completely devastated.
Mladenovic was one of several athletes placed under an "enhanced protocol plan" for being in contact with compatriot Benoit Paire, who tested positive for the novel coronavirus and was withdrawn ahead of the Grand Slam.
Mladenovic said despite "thirty negative tests" she was still treated like a prisoner and a criminal.
"We're living a nightmare here. I have only one desire and that is to regain my freedom," the 44th-ranked Mladenovic told reporters.
"The conditions are atrocious and if I had known that playing cards for 40 minutes with a player who tested positive, but ultimately negative, would have resulted in these consequences, I would never have set foot in this tournament."