The Premier League said it will defer "test events" with fans after the government's decision this week to cap the amount of spectators inside venues at 1,000.
With the new season starting this weekend there was hope that fans might be allowed back in stadiums, under strict health protocols, from Oct. 1 but that now looks unlikely.
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Talks took place over permission to use Manchester United's game against Crystal Palace on Sept. 19 as a test event and allow up to 12,000 supporters inside the stadium.
No fans have been allowed at Premier League games since last season was paused in March before a three-month hiatus caused by the ongoing COVID-19 pandemic.
The Premier League have written to the government to voice their disappointment at the situation provoked by a rise in coronavirus infections.
"With test events now capped at 1,000 supporters, we believe they will not provide any opportunity to properly test and evaluate measures designed to maximise fan safety," the statement read.
"Therefore, Premier League clubs will defer holding test events until a sufficient number of fans are allowed back to enable thorough trials to take place.
"At 1,000 supporters, not only would there be little to learn from a test event, but each match would be heavily loss-making."
Earlier this week, Premier League chief Richard Masters said it was "absolutely critical" for fans to return. He predicted the 20 clubs would lose a combined £700 million if fans are barred from stadiums all season.
Brighton & Hove Albion allowed 2,500 fans to their preseason friendly against Chelsea and the Premier League believe clubs are set up to provide safe environments.
"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 statement added.