Marketa Vondrousova's first defence of her Wimbledon title ended at the first hurdle when she was beaten in straight sets by Jessica Bouzas Maneiro on Tuesday.
Meanwhile, Novak Djokovic cruised to an easy win just 27 days after a knee operation but Andy Murray was forced to pull out of the singles tournament following his recent back surgery.
Czech sixth seed Vondrousova was a shock winner at the All England Club last year when she became the first unseeded woman to claim the trophy at the grass-court Grand Slam tournament.
But Vondrousova – who arrived in London as a fitness doubt after injuring her right hip in a fall at the Berlin tournament last month – was thrashed 6-4, 6-2 by world No 83 Bouzas Maneiro on her return to Centre Court.
German legend Steffi Graf was the last defending champion to lose in the opening round, when she was beaten by Lori McNeil in 1994.
Until Tuesday, the Spaniard had never defeated a top-40 player, while her only other grass-court match this year was a first-round loss in a low-key event in Italy.
“This is one of the most important moments in my life and career,” said Bouzas Maneiro. “She is one of the best players in the world so there was no pressure for me. I could just enjoy the moment and play freely.”
The 21-year-old added: “I'm really surprised with myself, honestly. At the beginning I was a little bit nervous but then after the first game the atmosphere was so nice and I was comfortable here, like I was at home.”
There were no such problems for fourth seed and 2022 champion Elena Rybakina, with the Kazakh easing past Romania's Elena-Gabriela Ruse 6-3, 6-1 on Court One. Fifth-seeded American Jessica Pegula is also through after demolishing countrywoman Ashlyn Krueger 6-2, 6-0 on Court Two.
Meanwhile, Andy Murray's hopes of being fit for singles action at this year's tournament are over after the two-time champion failed to recover in time from back surgery.
The 37-year-old was desperate to make one final singles appearance on his beloved Centre Court but admitted defeat ahead of his first-round match against Tomas Machac of the Czech Republic.
Murray, who secured the title in 2013 and 2016, underwent surgery to remove a cyst from his spine last month after being forced to pull out of Queen's, a procedure he described as “not insignificant”. The cyst was compressing his nerves making Murray lose control and power in his right leg.
Despite the latest setback in what has been an injury-ravaged last few years in his career, the three-time Grand Slam winner – who also won the 2012 US Open – was determined to give himself as much chance as possible of enjoying a singles farewell at SW19.
But Murray ran out of time for that, although the former world No 1 will still play doubles with his brother Jamie in what is bound to be an emotional farewell in front of his adoring British fans. He was replaced in the draw by Belgium's David Goffin, a lucky loser from qualifying.
“It’s probably a few days too soon. But I worked extremely hard to at least give myself a chance to play. It was the right decision,” said Murray on Tuesday afternoon.
“It is extremely disappointing that I wasn’t able to play but, at the same time, where I’m at 10 days after the operation in comparison to where I was told I would be and what my expectations were is incredible really.
“I obviously practised with my brother today and I look forward to playing the doubles.”
Murray, who ended a 77-year wait for a British men's singles champion at the London Slam, has a Wimbledon record of 61 wins and 13 defeats which puts him ninth in the all-time list, between Rafael Nadal and Jimmy Connors.
He is on record saying that this year is set to be his last on the court and could well bow out at this summer's Olympic Games. The tennis competition in Paris begins on July 27.
In the men's draw on Tuesday, Djokovic proved once again that he should never be written off after cruising to victory on Centre Court just 27 days after a knee operation.
The 37-year-old’s chances of featuring at Wimbledon looked all but impossible when he went under the knife in Paris last month for surgery on a torn meniscus.
But a 6-1 6-2 6-2 first-round stroll against Czech qualifier Vit Kopriva demonstrated to the doubters that not only is Djokovic fit, but he is also determined to at least challenge for title number eight at the All England Club.
Afterwards, Djokovic said: “I’m very pleased with the way I felt on the court. Obviously coming into this year was different circumstances because of the knee, and didn’t know how everything would unfold on the court. I’m extremely glad about the way I felt and the way I played.
“I tried to really focus on the game and not think too much about the knee. I’ve done everything possible in the last three and a half weeks. If it was any other tournament I probably wouldn’t have risked it – but I love Wimbledon.”
Polish seventh seed Hubert Hurkacz rallied from a set down to beat Moldova's Radu Albot 5-7, 6-4, 6-3, 6-4.
