The Premier League confirmed on Tuesday that 40 players and staff tested positive for coronavirus in two rounds of testing over the past week but insisted the season would continue as planned.
That figure is more than double the previous record of 18 positive cases and comes as England enters a nationwide lockdown to halt soaring infection rates.
Three matches were postponed last week due to outbreaks of Covid-19 at Manchester City and Fulham.
However, the British government has given the go-ahead for elite sport to continue despite the tighter restrictions.
Twice-weekly testing, used during the Project Restart plan to complete last season, has resumed in the Premier League after being scaled down to once-a-week at the start of the season.
In the first round of testing last week, 28 positive cases were detected from 1,311 tests, with a further 12 positives from 984 tests in the second round of testing.
Despite the postponements, rising case numbers and calls from some within the game for a circuit breaker to buy time and bring infection rates down, the Premier League said it remained confident the season could proceed as planned.
-With low numbers of positive tests across the overwhelming majority of clubs, the league continues to have confidence in its Covid-19 protocols, fully backed by the government, to enable fixtures to be played as scheduled- the league said in a statement on Tuesday.
The packed fixture schedule, with domestic leagues, cup and European competitions having to be finished before the delayed Euro 2020 starting on June 11, leaves little room for manoeuvre in the calendar.
A number of embarrassing incidents have come to light in which which prominent players have flouted coronavirus restrictions.
Pictures of Tottenham trio Erik Lamela, Sergio Reguilon and Giovani Lo Celso and West Ham's Manuel Lanzini attending a Christmas party with family and friends emerged over the weekend, prompting Spurs to promise internal disciplinary proceedings.
City have also launched an investigation after left-back Benjamin Mendy admitted to breaching Covid protocols by hosting a New Year's Eve party.
Reguilon and Mendy were included on the bench for Tottenham and City over the weekend, leading to calls for stricter sanctions against players who put the future of the season at risk by breaking the rules.
Despite City's issues with the virus, which left them without four first-team regulars for Sunday's 3-1 victory at Chelsea, manager Pep Guardiola called for more understanding for players.
-It would be better if, before we judge others, we judge ourselves- said Guardiola. -I'm not justifying that, he broke the rules. But don't give too many lessons to others. -
-I don't think footballers are more important than doctors, architects or teachers for my kids. So, they only have the same responsibility as everyone else in the world. -
-Mendy knows he made a big mistake, but it finishes here.-