Osaka, 23, was one of the faces of the Games in the Japanese capital and had the honor of lighting the Olympic trophy at the opening ceremony last week.
But the four-time Grand Slam champion could not prove that by winning the medal, losing 6-1 6-4 to the world number 42 in the third round.
It was Osaka’s first tournament after withdrawing from the French Open in June, where he revealed he had “had a long period of depression” since winning his first Grand Slam title in 2018.
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Following an impressive win in the first two rounds, Osaka looked to be a good gold medal bid but produced 32 uncontrollable errors in rough action against former French Open finalist Vondrousova.
The 22-year-old Czech midfielder has beaten 18 winners in 10 uncontrolled errors as he has converted five of his 11 points in the match.
Osaka’s exit follows Australia’s top-seeded Ashleigh Barty in the first round, leaving women’s songs very open.
Elina Svitolina, a fourth-generation Ukrainian star, is the top performer left in the competition.
Vondrousova will play either Paula Badosa of Spain or Nadia Podoroska of Argentina in the last eight.