Nature Valley International top seed Guido Pella slumped out in his first match of the tournament, while Benoit Paire suffered a similar fate at the Antalya Open.
Brasil Open winner Pella was handed a bye in the first round at Eastbourne, but came undone against unseeded American Taylor Fritz on Wednesday.
Fritz let his Argentine opponent back into the contest after cruising in the first set, but Pella's resistance was not enough as he went down 6-4 3-6 6-4.
Fellow seeds Laslo Djere and Dusan Lajovic also dropped out, losing 7-6 (7-5) 7-6 (8-6) and 6-2 6-3 to Thomas Fabbiano and Sam Querrey respectively.
The No. 1 seed is out at the #NatureValleyInternational @Taylor_Fritz97 defeats Pella 6-4, 3-6, 6-4 to make another #ATPTour quarter-final in 2019
— ATP Tour (@ATP_Tour) June 26, 2019
: @TennisTV | pic.twitter.com/GkkgMeeeWY
Though the top four seeds are now all out, Fernando Verdasco – who needed three sets to see off Juan Ignacio Londero – and Gilles Simon, fresh from finishing as runner up at the Queen's Club Championships, both progressed, and will be among the favourites heading into the closing days of play.
Dan Evans also eased through, beating Pierre-Hugues Herbert 6-3 7-5, while Kyle Edmund overcame Cameron Norrie 6-2 6-2 to set up an all-British quarter-final.
The top seed was also beaten at the Antalya Open on Wednesday, as qualifier Viktor Troicki recorded a surprise win over world number 32 Paire.
Former world number 12 Troicki missed three months of last season due to a back injury, and entered the tournament way down the ATP rankings, but shocked Paire with a 7-6 (7-4), 7-6 (7-5) win.
Troicki will next play Miomir Kecmanovic, who knocked out sixth seed Ugo Humbert 6-4 7-5, in the last 16.
Fourth seed Pablo Carreno Busta edged past Kevin Krawietz, and Bernard Tomic overcame Peter Gojowczyk in Wednesday's other matches.
Top Headlines
Featured Matches
Football
Arsenal
English Premier League
2-0
Brighton and Hove Albion
Aston Villa
English Premier League
2-0
Chelsea
Fulham
English Premier League
0-1
Newcastle United
Leeds United
English Premier League
2-0
West Bromwich Albion
Leicester City
English Premier League
2-1
Tottenham Hotspur
Manchester City
English Premier League
2-0
Everton
Liverpool
English Premier League
1-0
Crystal Palace
Cricket
Sussex
Sri Lanka
Teams will be announced at the toss
Multan Sul..
Quetta Gla..
Teams will be announced at the toss
Islamabad..
Karachi Ki..
Teams will be announced at the toss
Peshawar Z..
Lahore Qal..
Teams will be announced at the toss
Islamabad..
Quetta Gla..
Teams will be announced at the toss
Lahore Qal..
Islamabad..
Teams will be announced at the toss
Mumbai Ind..
Delhi Capi..
Teams will be announced at the toss