Dallas Cowboys running back Ezekiel Elliott will immediately begin a six-game suspension after the NFLPA's request for an emergency injunction on his behalf was denied.
Judges at the US Second Circuit of Appeals denied Elliott's appeal, saying he "failed to meet the requisite standard".
Elliott is expected to appeal and the court has ordered that any such appeal be heard on "an expedited basis" but he now appears certain to miss Sunday's game against the Atlanta Falcons.
The decision to not grant a preliminary injunction comes after a lower court denied the same request last month, reinstating Elliott's suspension briefly before a stay was issued by the 2nd Circuit.
If Elliott's six-game ban holds up, he will also miss games against the Philadelphia Eagles, Los Angeles Chargers, Washington Redskins, New York Giants and Oakland Raiders. He would not be eligible to return until Week 16 against the Seattle Seahawks on December 24.
Elliott has been embroiled in a legal saga with the NFL after being issued a six-game suspension following a 13-month investigation into a domestic violence allegation in 2016.
Although Elliott faced no criminal charges from the case, the NFL probe found him to have been in breach of the league's personal conduct policy.
Elliott has bitterly contested the ban and received an administrative stay from the New York federal court last Friday, reinstating his eligibility for Sunday's game against the Kansas City Chiefs.
He led the Cowboys in rushing with 93 yards on 27 carries and scored the go-ahead touchdown in the 28-17 win after getting another reprieve.
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