Danny Rose joined Alex McCarthy in pulling out of the England squad through injury, meaning Ben Chilwell is in line to make his first senior start in Friday's Nations League clash with Croatia.
Three months after facing one another in the World Cup semi-finals, the Three Lions travel to Rijeka before heading onto an arguably even tougher assignment against Spain.
Southgate will be without Tottenham left-back Rose and Southampton goalkeeper McCarthy for those games after withdrawing from the squad on Thursday due to the "minor injuries" they arrived with.
While England still have three others goalkeepers to choose from, it leaves Leicester's Chilwell as the only left-back in the squad travelling to Croatia.
The 21-year-old got his first chance last month and came off the bench against Switzerland after being called up as a replacement for Manchester United's Luke Shaw - the same player he replaced in the squad on Sunday.
Brighton's Lewis Dunk has been the other late call-up following an injury to James Tarkowski of Burnley, but there does not appear to be plans for fresh faces before the Croatia game.
Danny Welbeck, another who had sat out training earlier in the week as a precaution, was also missing again as England trained at St George's Park on Thursday morning, but the Arsenal forward was set to travel with the squad to Croatia in the afternoon.
The other 22 members of Southgate's group were involved in the session.
Friday's match will be played behind closed doors at Stadion HNK Rijeka as Croatia complete a stadium ban imposed by UEFA for having a swastika on the pitch in a Euro 2016 qualifier against Italy.
England then take on Spain three days later in Seville.
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Odds correct as of 1901 BST on 10/10/18