Pablo Carreno Busta headlined the casualties at the Winston-Salem Open, where the top seed was shocked in straight sets by Ilya Ivashka.
The 2016 champion, Carreno Busta had his sights set on the semi-finals of the ATP 250 tournament but he suffered a surprise 7-6 (7-2) 6-3 loss on Thursday.
"Of course it's a great feeling. I had a great match today. These conditions suit me," Ivashka said. "I think he had more pressure because he's the top seed so for me it was a good challenge to see what my level is and to compete with these guys, so for me it was a great match."
Carreno Busta – who trumped world number one and 20-time grand slam champion Novak Djokovic to win bronze at the Olympic Games – was not the only seed to fall.
Richard Gasquet and Frances Tiafoe also crashed out in the quarter-finals following defeats to Emil Ruusuvuori and Mikael Ymer.
The only seed to advance to the semis was 15th seed Carlos Alcaraz, who saw off Marcos Giron.