World number three Maria Sakkari was knocked out of the US Open in the second round by China's Wang Xiyu.
Greece's Sakkari, who reached the semi-finals in New York last year, won the first set but could not capitalise on her lead, losing 3-6 7-5 7-5.
It is the third time Sakkari has lost to a player ranked outside the world top 50 at a Grand Slam this year.
Wang, meanwhile reaches the third round of a major for the first time.
There were fewer second-round troubles for world number five Ons Jabeur, however, as she battled to a straight-sets win over American Elizabeth Mandlik.
Tunisian Jabeur, who has never progressed past the third round at Flushing Meadows, won 7-5 6-2 though saw Mandlik twice go up a break and had to save a set point in the first set.
Home hope Coco Gauff, seeded 12th, also reached the third round as the 18-year-old beat Romania's Elena-Gabriela Ruse 6-2 7-6 (7-4).
Gauff will face compatriot Madison Keys next, with American Shelby Rogers playing Wimbledon finalist Jabeur in the third round after beating Slovakia's Viktoria Kuzmova 7-5 6-1.
Runner-up in 2017, Keys, who is the 20th seed in New York and was victorious against world number one Iga Swiatek earlier in August, won her second-round match against Italy's Camila Giorgi 6-4 5-7 7-6 (10-6).
But Canada's Leylah Fernandez, who lost last year's US Open final to Emma Raducanu, is out after she was beaten 6-3 7-6 (7-3) by Russian Ludmilla Samsonova.
She will face Aleksandra Krunic in the third round, the Serb coming from behind to beat 23rd seed and 2021 French Open champion Barbora Krejcikova in three sets.
Canada's Bianca Andreescu, who won the US Open in 2019, put in an impressive performance to earn a 6-2 6-4 victory over Brazil's Beatriz Haddad Maia and will next play Caroline Garcia, of France.