French Open champion Garbine Muguruza and Wimbledon runner-up Milos Raonic were knocked out of the US Open on Wednesday.
Muguruza slumped to a 7-5, 6-4 loss to Latvia's Anastasija Sevastova, the world number 48.
Having not won a match at the US Open in five years before this week, 26-year-old Sevastova now goes on to face Kateryna Bondarenko, a 5-7, 7-6 (7/5), 7-5 winner over China's Zheng Saisai.
Meanwhile, world number one Novak Djokovic made the last 32 of a major for the 33rd successive time without hitting a ball when Jiri Vesely of the Czech Republic withdrew from their second round encounter suffering an inflammation of the left forearm.
Djokovic will face 34-year-old Russian Mikhail Youzhny, twice a semi-finalist, on Friday for a place in the last 16.
Also, 120th-ranked Ryan Harrison of the United States stunned fifth seeded Raonic 6-7 (4/7), 7-5, 7-5, 6-1 in another second round upset.
Harrison has now reached the third round. He now goes on to face experienced Cypriot Marcos Baghdatis for a place in the last 16.
Former number one Caroline Wozniacki, the US Open runner-up in 2009 and 2014, defeated 2004 champion Svetlana Kuznetsova 6-4, 6-4.
Britain's Johanna Konta, the 13th seed, collapsed on court with heat sickness before completing a 6-2, 5-7, 6-2 win over Bulgaria's Tsvetana Pironkova.
Britain's Kyle Edmund backed up his first round win over world number 15 Richard Gasquet with a 7-5, 6-4, 6-4 victory over US wildcard Ernesto Escobedo on the back of 53 winners.
John Isner of the United States fired 38 aces to beat Belgian qualifier Steve Darcis 6-3, 6-4, 6-7 (10/12), 6-3 and next faces Edmund.
Croatian seventh seed Marin Cilic, the 2014 champion, made the last 32 by seeing off Sergiy Stakhovsky 6-1, 6-2, 6-3.
Later Wednesday, Spanish fourth seed Rafael Nadal, the 2010 and 2013 champion, takes a 7-1 career record over Italy's Andreas Seppi into their night time clash.