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Briton Joe Salisbury and American partner Rajeev Ram suffered their first loss at the US Open since 2020 as the three-time defending champions' winning run at Flushing Meadows ended.
The men's doubles winners in 2021, 2022 and 2023 were beaten 7-6 (7-3) 6-3 in the third round by American 13th seeds Nathaniel Lammons and Jackson Withrow.
Salisbury and Ram's winning record in New York stretched back 20 matches, with their most recent defeat before Monday coming in the 2020 semi-finals.
The pair also won the Australian Open in 2020, in addition to their three other Grand Slam titles at the US Open.
They have struggled at the major events since winning their third US Open last year, however, exiting the Australian Open in the third round in January, losing in the quarter-finals of the French Open and falling to a second-round upset at Wimbledon.
Wimbledon champion and fellow Briton Henry Patten and partner Harri Heliovaara were also knocked out in round three, losing 6-2 6-2 to 11th seeds Wesley Koolhof and Nikola Mektic.
Britain's Harriet Dart and French partner Diane Parry were also beaten in the third round of the women's doubles.
Having beaten Olympic champions Jasmine Paolini and Sara Errani in round two, Dart and Parry lost 6-4 6-4 to 10th seeds Hao-Ching Chan and Veronika Kudermetova.