After being dropped for last month’s 0-0 draw against Slovenia, Rooney made a return to Gareth Southgate’s starting XI for their World Cup qualifier with Scotland.
The 31-year-old played the full 90 minutes of Friday’s game, setting up Gary Cahill for England’s third goal.
Bertrand, who started last month’s 2-0 win against Malta, was unused by Southgate but both are now doubts for the game with Spain.
The Football Association confirmed that the two senior players would sit out Monday’s session but did not indicate if either will be withdrawn from Tuesday’s match-day squad.
In their absence, Tottenham youngster Dylan Duncan will train with the senior side for the first time.
Meanwhile, Southgate confirmed yesterday that Tottenham striker Harry Kane would return to the north London club from international duty.
Like Bertrand, Kane remained an unused sub during England’s 3-0 victory over Gordon Strachan’s side.
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