Williams, playing her first singles match in four years, was beaten by Australia’s Maya Joint in a three-set epic on Centre Court late on Tuesday evening.
The 44-year-old, who retired in 2022, made a sensational comeback in June, playing doubles tournaments at Queen’s Club and Berlin.
She is due to join forces with sister Venus Williams in the women’s doubles first round against Camila Osorio and Solana Sierra.
There had been no indication of any injury to the 23-time Grand Slam singles champion during the Joint loss.
But Serena did not attend the mandatory post-match press conference, instead issuing a short quote through tournament organisers.
Players can be fined for skipping media duties but it was revealed on Wednesday that Williams is dealing with a knee problem.
A statement from the seven-time Wimbledon singles champion’s agent Jill Smoller read: “Serena tweaked her right knee at the end of the first set and was therefore excused from her media obligations by the Wimbledon and WTA medical teams.
“She left site that night unaided and is doing everything she can to be ready for her doubles match later this week.”
Serena and Venus, 46, who were given a wildcard entry into the tournament, have won the Wimbledon doubles six times, with their most recent title coming in 2016.
Serena has not won a Grand Slam singles crown since the 2017 Australian Open, while Venus last lifted a major singles trophy at Wimbledon in 2008.
Serena said her astonishing comeback was motivated by a desire to play in front of her two young daughters.
Her children watched their mother’s loss to Joint alongside her husband Alexis Ohanian and Venus in the players’ box on Tuesday.
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