Holmes was sentenced to just over 11 years in prison for defrauding investors with her Silicon Valley start-up Theranos.
She was scheduled to begin serving prison time on April 27, but her lawyers lodged a last-minute appeal on procedural issues after an earlier attempt was denied.
US Judge Edward Davila on Tuesday denied the new request and in a separate ruling ordered Holmes and her top Theranos lieutenant, Ramesh “Sunny” Balwanito, to pay $452 million to victims of their fraud.
According to media reports, Davila has not yet decided on the new date of her imprisonment and has recommended that she serve her sentence at a women’s prison in Texas.
Holmes was found responsible of duping investors into believing that she had developed a revolutionary medical device.
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The 39-year-old became a star of Silicon Valley when she said her start-up was perfecting an easy-to-use test kit that could carry out a wide range of medical diagnostics with just a few drops of blood. But her company flamed out after a Wall Street Journal investigation into the validity of the tests.
Holmes had a child shortly before her trial and has had a second since her conviction.