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The English Football League says a quarter of players at its member clubs "do not intend to get a vaccine" against Covid-19.
Tighter protocols at first-team training grounds and more regular testing have been enforced.
It follows the postponement of more fixtures scheduled to be played on Saturday across all three divisions.
Updated guidance on the minimum number of available players required to fulfil a fixture has also been passed on.
Clubs will be expected to play a fixture where they have 14 players, including a goalkeeper, available from the their registered squad list.
Vaccine data collected by the EFL in November showed 75% of players were either fully vaccinated, had had a single jab or intended to be vaccinated.
Double-vaccinated players totalled 59%, with 16% set to get either a second or first jab at the time.
In September, 49% of players in the EFL were double vaccinated, which was up from 18% the previous month.
The EFL's medical adviser, Dr Richard Higgins stressed they are continuing to "strongly encourage players and staff at clubs to get fully vaccinated and obtain a booster jab if eligible to do so".
He added: "We know getting double jabbed and boosted now will reduce the risk of getting seriously ill or dying from Covid-19, reduce the risk of catching or spreading Covid-19 amongst team-mates and loved ones, while helping protect against variants including Omicron."
As fixture postponements have increased over the past week, the EFL has also confirmed all 72 clubs will need to implement their set of "red" protocols to try to mitigate the spread of infection in first-team training facilities.
More to follow.
Saturday's postponed EFL matches
Championship
Cardiff City v Derby County
Coventry City v Stoke City
Millwall v Preston
QPR v Swansea City
Reading v Luton Town
League One
Bolton Wanderers v Wycombe Wanderers
Gillingham v Crewe Alexandra
Lincoln City v Doncaster Rovers
Sheffield Wednesday v Accrington Stanley
League Two
Crawley Town v Oldham Athletic
Northampton Town v Barrow
Swindon Town v Walsall
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