Venus, a five-time champion at the All England Club who last lifted the trophy in 2008, then lost for the second year running to Bulgarian number 32 seed Tsvetana Pironkova, 6-2 6-3 this time.

The last time both Williams sisters failed to reach the quarter-finals was in 2006, when Amelie Mauresmo went on to win the title.

“This was definitely not our best day. I think we both envisioned seeing this day going a little bit different,” said Venus.

Wimbledon’s top seed and world number one Caroline Wozniacki also bowed out on a day of big-name casualties, losing a nailbiting clash to Dominika Cibulkova 1-6 7-6 (7/5) 7-5.

Tomorrow Cibulkova will face 2004 champion and firm title favourite Maria Sharapova, who reached the quarter-finals with a 6-4 6-2 victory over Shuai Peng.

Bartoli’s prize for her first career victory over Serena Williams is a last-16 meeting with Sabine Lisicki, who beat Petra Cetkovska 7-6 (7/3) 6-1.

Pironkova will face Czech eighth seed Petra Kvitova, who breezed through with a 6-0 6-2 victory over Belgian Yanina Wickmayer.

And fourth seed Victoria Azarenka will go up against the world number 80 Tamira Paszek for a place in the semi-finals after brushing past Nadia Petrova 6-2 6-2. Austrian Paszek beat Ksenia Pervak of Russia 6-2 2-6 6-3.

Both Williams sisters have had their year disrupted by injury and they arrived at Wimbledon without any form and only limited preparation at Eastbourne.

Serena had played just two matches all year before her first-round tie after recovering from blood clots on her lung.

Her defeat makes this only the third time in the open era that the defending women’s champion has failed to make the last 16 at Wimbledon.

“I obviously would have loved to do better but considering the condition I was in, considering a lot of things, I really feel I did well,” said Williams.

“I would have been sad being at home watching it on TV, like I’m going to be soon.

“I made an effort to get out here and play a couple of tournaments. I just can’t sit here and be disappointed. For the most part, I can just use this as momentum going forward.

“I can only get better and that can potentially be really scary, because I can only go up from here and I can just do so much more.”

Serena, a 13-time grand slam champion, will now plummet to 180th in the world and will require a wild card to compete at the US Open, unless she can earn some ranking points during the first part of the hard court season.

Venus, who had also only returned to action at Eastbourne after suffering a hip injury at the Australian Open in January, was comprehensively outplayed by Pironkova.

The repeat of last year’s quarter-final resulted in an identical outcome as the highly impressive Pironkova, 23, made light work of Williams.

Wozniacki, world number one at the age of 20 despite not having a grand slam title to her name, looked in charge when she won the opening set of her match in a hurry but 24th seed Cibulkova turned the match around to win.

Sharapova had lost her position as title favourite to Serena Williams over the weekend – but she reclaimed it today.

The Russian fifth seed has yet to drop a set and she has reached the Wimbledon quarter-finals for the first time since 2006.