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The US Open women's draw has lost its top two seeds after Caroline Wozniacki suffered a shock second-round loss to Ukraine's Lesia Tsurenko.
The world number two from Denmark has reached the US Open final twice but lost 6-4 6-2 to the world number 36.
World number one Simona Halep lost in the first round while Australian Open champion Wozniacki's defeat by Tsurenko was her third early exit this month.
"She played the game I was supposed to play," said Wozniacki, 28.
"This part of the season is usually a part of the season I really look forward to, one where I really play well."
Meanwhile, fourth seed and former champion Angelique Kerber overcame a second-set wobble against Sweden's Johanna Larsson to make the last 32.
The German, who had never lost a set in three previous matches with Larsson, eventually won 6-2 5-7 6-4.
Kerber plays Dominika Cibulkova next after the Slovak advanced despite a two-point deduction for returning late from a heat break.
Temperatures at Flushing Meadows were once again high enough for organisers to apply their extreme heat policy, but Cibulkova was deemed to have taken longer than the permitted 10 minutes to cool down between the second and third sets in her match against Taiwan's Hsieh Su-wei.
"[The umpire] told me I was one minute late. I'm getting two points' penalty. You cannot be serious," said Cibulkova, who won 7-6 (7-3) 4-6 6-4.
"It took me, I think, more than three minutes walking through the people, because there were so many fans and so many people. I couldn't believe it."
American Madison Keys, who lost last year's final to Sloane Stephens, beat compatriot Bernarda Pera 6-4 6-1 to set up a third-round match with Victoria Azarenka.
Fifth seed Petra Kvitova got past China's Wang Yafan 7-5 6-3, breaking late to seal the opening set after initially letting a 4-1 lead slip.
Kvitova, 28, next plays 20-year-old Belarusian Aryna Sabalenka, who won the New Haven title earlier in August.
Sabalenka beat 2010 runner-up Vera Zvonareva 6-3 7-6 (9-7) to reach the last 32.
She is joined by her compatriot Aliaksandra Sasnovich, who upset Russia's in-form 11th-seed Daria Kasatkina 6-2 7-6 (7-3)
Sasnovich will face Naomi Osaka - who clinched her biggest career title at Indian Wells in March - after the Japanese beat Julia Glushko 6-2 6-0.
