Tomas Berdych was the highest profile casualty at the Western and Southern Open on Wednesday, falling to Lu Yen-Hsun.
It was the Chinese Taipei man's first win over Berdych in four career meetings, all of which have been played on hard courts.
Lu prevailed 3-6 6-3 6-4 over the Czech fourth seed at the ATP 1000 event, the world number 43 reversing his loss to Berdych at the Rogers Cup in Toronto earlier in August.
Other seeds to crash out in Ohio included Grigor Dimitrov (seven), Ernests Gulbis (nine) and Roberto Bautista Agut (13), with big names Roger Federer and Andy Murray safely through to the last 16.
Swiss second seed Federer won his 300th career match at a Masters-level tournament, when he saw off Canada's Vasek Pospisil 7-6 (7-4) 5-7 6-2.
Eighth seed Andy Murray had less trouble getting past Portugal's Joao Sousa, the Briton a 6-3 6-3 winner.
Canadian fifth seed Milos Raonic downed American wildcard Robby Ginepri 6-2 6-2, while sixth seed David Ferrer of Spain endured an almighty struggle to beat German Philipp Kohlschreiber 6-7 (4-7) 7-6 (7-4) 7-6 (7-4).
Bulgaria's Dimitrov was outgunned by Poland's Jerzy Janowicz 6-4 3-6 6-3, while Latvian Gulbis was no match for American wildcard Steve Johnson 6-4 6-4.
Frenchman Gael Monfils dumped out Spaniard Bautista Agut 6-4 6-1.
American number one and 11th seed John Isner progressed with a 6-3 7-6 (7-1) win over Australian qualifier Marinko Matosevic.
Matosevic's compatriot Lleyton Hewitt joined him in a second-round exit, the veteran beaten by Italian 15th seed Fabio Fognini 6-1 6-4.
Spanish 16th seed Tommy Robredo dispatched of American wildcard Sam Querrey 6-2 6-4, while Marin Cilic - the 14th seed from Croatia - advanced past Spain's Fernando Verdasco in a break-free contest 7-6 (8-6) 7-6 (7-5).
France's Julien Benneteau saw off British qualifier James Ward 6-2 6-2, and Russia's Mikhail Youzhny defeated Italy's Andreas Seppi 6-3 6-4.
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