Venus Williams was unable to win back-to-back WTA Tour matches for the first time since 2019 after letting a lead of a set and a break slip in Cincinnati.
On the day Williams received a wildcard for a record-extending 24th US Open, the 43-year-old American lost 1-6 6-2 6-1 to world number 24 Zheng Qinwen.
Williams, ranked 533rd, led 6-1 2-0 before losing 11 games in a row.
China’s Zheng, 20, served out victory and plays top seed Iga Swiatek in the Western and Southern Open last 16.
Williams will now continue her preparations for the US Open, where she will extend her own record of Open era singles appearances.
The seven-time major champion made her main-draw debut at Flushing Meadows in 1997, going on to win the 2000 and 2001 titles.
Two-time finalist Caroline Wozniacki, who is making a comeback after retiring in 2020, is also among the wildcards.
Dane Wozniacki, 33, will play in her third tournament since returning to the WTA Tour earlier this month.
The US Open, which is the final major event of the season, starts on 28 August.
Swiatek looking sharp before US Open defence
Polish world number one Swiatek, 21, continued her preparations for the defence of her title in New York with a ruthless 6-1 6-0 win over American wildcard Danielle Collins.
“I feel like I was really solid and I didn’t make a lot of unforced errors,” Swiatek said. “I really like playing that way because it gives me confidence.”
American world number three Jessica Pegula, who won the Canadian Open title last week, also reached the Cincinnati last 16 with a 6-7 (2-7) 6-2 6-3 victory over Italy’s Martina Trevisan.
Wimbledon champion Marketa Vondrousova of the Czech Republic won 6-4 6-2 against Russia’s Anastasia Popatova, while Russian 14th seed Daria Kasatkina also progressed by beating France’s Varvara Gracheva.
Greek eighth seed Maria Sakkari avoided a shock against Romania’s Sorana Cirstea, fighting back to win 2-6 6-3 7-5.
Djokovic ‘already over’ Wimbledon defeat
Serbia’s Novak Djokovic plays his first match since losing the Wimbledon final to Carlos Alcaraz when he takes on Spain’s Alejandro Davidovich Fokina in Wednesday’s night session (00:00 BST, Thursday).
“It’s not the first [nor] the last match that I lost, so I was over it in a day,” 23-time major champion Djokovic, who has not played in the US since 2021 because he was not vaccinated against Covid-19, said.
“I need to obviously move on and try to regroup and find motivation for what’s coming up, and all the challenges that are ahead of me, and that’s why I’m here.”
Earlier in the men’s singles, Russian third seed Daniil Medvedev moved into the last 16 with a 6-3 6-2 win over Italy’s Lorenzo Musetti.
Medvedev, who claimed the title in 2019 before going on to reach the US Open final, faces Germany’s Alexander Zverev next.
Zverev won 7-5 6-4 against Japan’s Yoshihito Nishioka, while Greek fourth seed Stefanos Tsitsipas and American ninth seed Taylor Fritz both also progressed.
Tsitsipas plays Poland’s Hubert Hurkacz, who knocked out Croatian defending champion Borna Coric.
But there were shock defeats for Norwegian fifth seed Casper Ruud and Italian eighth seed Jannik Sinner.
Last year’s US Open finalist Ruud lost to Australia’s Max Purcell, with Toronto champion Sinner going out to Serbia’s Dusan Lajovic.