The Premier League has written to the government to express concern at the "decisions taken to restrict and potentially delay" the return of fans to football stadiums.
On Wednesday, Prime Minister Boris Johnson said plans for spectators to attend sporting events in England from 1 October are to be reviewed after a rise in coronavirus cases.
Pilot events in September will be restricted to 1,000 fans, but the Premier League says it will not hold test events until more can attend.
In a statement, it said the 1,000-person cap will "not provide any opportunity to properly test and evaluate measures designed to maximise fan safety".
"At 1,000 supporters, not only would there be little to learn from a test event, but each match would be heavily loss-making," the league said.
"For each month of the season without fans, more than £100m is lost to football across the leagues, with consequent damage to the local and national economy.
"It also harms the employment prospects of more than 100,000 people whose jobs depend on matchday activities."
Rugby Football League chief executive Ralph Rimmer has also called on ministers to reconsider plans to delay the return of fans, claiming clubs in his sport could be hit with losses of "up to £2m per week".
At the end of August, 2,500 people watched a friendly between Brighton and Chelsea at the Seagulls' Amex Stadium - the first time fans had been allowed into a Premier League ground in almost six months.
Football has been played behind closed doors since it resumed in June following the coronavirus lockdown.
Premier League chief executive Richard Masters has told BBC Sport it is "absolutely critical" fans are allowed back inside stadiums as soon as possible and that failure to do so would cost clubs £700m during the 2020-21 season, which starts on Saturday.
"Our clubs have already prepared test events and can offer larger scale trials to prove it is possible to produce bio-secure, safe environments, as was proved through the successful delivery of Project Restart," the Premier League added.
"The Premier League recognises the ambition of government's 'Operation Moonshot' and will support the project's objectives to get fans back into stadiums. However, we believe measures are already available which would allow the safe return of fans and should also be activated now.
"As the government begins its review of the 1 October date for the return of spectators to sport, the Premier League asks that the high safety standards which can be achieved by our clubs are fully taken into account before any decision is taken and that sport is not left behind other industries."
Meanwhile, it was announced on Friday that Premier League executive director Bill Bush, who has been key in negotiations between the organisation and the government over the return of football with fans, is leaving his role.
Bush will remain as an adviser to the board and the chief executive.