Captain Leah Williamson and defender Lucy Parker have joined the growing list of stars hit with injuries ahead of this Friday's match, which is set to be played in front of a sell-out Wembley crowd.
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Williamson - who also wears the armband for current WSL leaders Arsenal - will 'undergo further assessment' after sustaining an injury in training.
This means she could still feature at Wembley, with her yet to return to Arsenal and still being with England as it stands.
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Meanwhile, West Ham defender Lucy Parker - who received her first call-up - has also suffered an injury, but will miss the game at Wembley and is set to return to the Hammers.
As a result, European champions Lotte Wubben-Moy and Nikita Parris have been called up after not making the original squad.
Williamson and Parker join England and Manchester United striker Alessia Russo in missing out on the highly anticipated fixture that will see the European champions face the reigning world champions.
The 23-year-old - who scored four goals at this summer's Euros, including that delicious back-heeled effort - has suffered a small injury and withdrew from the Lionesses squad on Monday.
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Sarina Wiegman's side will be hoping to put their injury woes aside.
Tickets for the friendly sold out in just 24 hours in August, days after England beat Germany to be crowned European champions for the first time.
Wiegman recognises how special it is for her team to be playing in front of such big crowds, too, telling talkSPORT: "It's so nice. We had an incredible summer, we'll take that with us for the rest of our lives.
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"But we also want to move on and it's so nice to see this momentum and that the fans are so enthusiastic."
The Lionesses are also set to face the Czech Republic in Brighton next Tuesday as they prepare for next summer's World Cup in Australia and New Zealand.