Sergio Ramos suffered an injury during the first half of the Nations League match between Spain and Germany in Seville.
The Real Madrid captain hurt his hamstring after playing a long pass forward and was replaced by Eric Garcia 43 minutes in.
Spain had already seen Sergio Canales go off with a similar complaint just 12 minutes into the contest.
Despite the injury issues, Luis Enrique's side led 3-0 at half-time in the decisive Group A4 match through goals from Alvaro Morata, Ferran Torres and Rodri.
Any injury to Ramos would be a major concern for Madrid head coach Zinedine Zidane ahead of a challenging run of fixtures
Madrid then face a further four games, including clashes with Shakhtar Donetsk, Sevilla and Borussia Monchengladbach, before the scheduled derby with Atletico Madrid on December 13.
Ramos missed the reverse fixture against Shakhtar on October 21, a match Madrid lost 3-2 after falling 3-0 down in the first half at Estadio Alfredo Di Stefano.
There was further bad news for Zidane in Paris too, as another of his centre-back options in Raphael Varane was withdrawn at half-time during France's meeting with Sweden.
Les Bleus boss Didier Deschamps told M6: "[Lucas] Hernandez and Varane have come off because they have small problems."