There will be no back-to-back Aegon International titles for Belinda Bencic after the defending champion was beaten by Elena Vesnina, and Roberta Vinci was among several seeds to crash out in the second round on Tuesday.
Bencic beat Agnieszka Radwanska to win the title in Eastbourne last year, but the third seed was dethroned when Elena Vesnina consigned her to a 7-6 (7-4) 7-6 (7-5) defeat.
World number eight Bencic withdrew from the Aegon Classic in Birmingham last week due to a thigh injury and suffered another blow just six days before Wimbledon commences.
The unseeded Vesnina was broken in the first game of the match, but hit straight back and also came from behind in the first tie-break in a match which saw both players fail to hold serve on three occasions.
Russian Vesnina, ranked 53rd in the world, managed to recover from being behind at 4-2 in the second set as she moved into round three at Devonshire Park.
Vinci also made an early exit, slumping to a 6-4 4-6 3-6 defeat to Vesnina's compatriot Ekaterina Makarova.
The 2015 US Open runner-up Vinci lost in the first round at the French Open and her disappointing form continued on the grass in Sussex, where she was broken eight times.
Top seed Radwanska was beaten by Coco Vandeweghe in the first round in her first appearance on grass this year, but the top seed reached the third round when Mirjana Lucic-Baroni retired due to illness.
The world number three was leading 6-4 2-1 when Lucic-Baroni was unable to continue, while two-time Wimbledon champion Petra Kvitova beat Timea Babos 6-4 7-6 (7-5).
Fourth seed Timea Bacsinszky was dumped out by Kristina Mladenovic, while Caroline Wozniacki easily ousted seventh seed Samantha Stosur 6-2 6-1.
Svetlana Kuznetsova was beaten by Kateryna Bondarenko, while Carla Suarez Navarro, Irina-Camelia Begu, Anastasia Pavlyuchenkova, Sara Errani and Lucie Safarova were the other players to bow out in round two.