Dallas Cowboys running back Ezekiel Elliott has lost an attempt to block his six-game ban, as a US judge rejected a request for a preliminary injunction.
US district court judge Katherine Polk Failla issued her ruling after hearing arguments from the league and the NFL Players Association (NFLPA) on Monday in New York.
Failla said in her ruling: "The NFLPA has failed to demonstrate a substantial question warranting the extraordinary remedy of injunctive relief or a balance of hardships that decidedly weighs in its favour.
"The arbitrator gave Mr Elliott ample opportunity, in terms of both proceedings and evidence, to challenge the Commissioner's decision."
Failla stayed the decision for 24 hours to allow the NFL Players Association time to consider appealing the ruling.
They have the option of appealing Failla's decision to the US Court of Appeals and seeking a stay that would allow Elliott to continue playing during the appeal process.
Elliott, 22, attended the court proceedings on Monday, completing a busy 24 hours.
One of the top running backs in the NFL, on Sunday night he had rushed for 150 yards and two touchdowns in a 33-19 win over the Washington Redskins.
Elliott was suspended for six games by NFL commissioner Roger Goodell on August 11 after a 13-month probe into allegations he assaulted an ex-girlfriend.
But Elliott, the fourth overall selection in the 2016 draft, was never arrested nor charged by police in the incident.
The players union was granted a preliminary injunction by a federal judge in Texas on September 8, but a federal appeals court in New Orleans sided with the NFL on October 12 and lifted the injunction.
The players union then went to the Southern District Court in New York.
The second-year running back was granted a temporary restraining order by district judge Paul Crotty on October 17, but that order is expiring.
While the case has been in court, Elliott has played seven games for the Cowboys this season.
However, if he now serves the six-game suspension, he would be ineligible until Dallas faces Oakland on December 17.