Arithmetical errors prevent from getting into the record books.
George Hood spent 85 hours riding a stationary bike in January, riding the equivalent of about 1,080 miles (1,738 kilometers), and thought he had bested the existing record of 82 hours.
However, Guinness World Records officials invalidated Hood's entry because of record-keeping errors.
About 40 volunteers took turns logging Hood's efforts, but they made addition and subtraction mistakes and had trouble reading a 24-hour clock, Guinness officials said.
The entry contained "fundamental flaws," said Guinness records manager Scott Christie.
"I'm more frustrated, not disappointed," said Hood, 49, a supervisor with the U.S. Drug Enforcement Administration.
Even without the record, his ride raised nearly $30,000 (22,122 EUR) for a group that helps families of police officers killed on duty.
And he is planning to try again this summer, hoping to hit 101 hours over four days.
"If I'm going to go through all this training and work, I want to be published," he said. "I won't be denied."