Vlaar, 29, came off just 24 minutes into Netherlands' 3-2 friendly loss to Mexico in Amsterdam.
Coach Guus Hiddink is unlikely to have the defender available for Sunday's Euro 2016 qualifier against Latvia.
Vlaar has been linked with moves to Arsenal and Manchester United.
"It does not look good," Hiddink said.
"Ron has problems with his calf. It is not the same as the last time, that place was totally healed."
Robin van Persie could return from a hamstring injury for the clash with the Dutch desperate for a win after just one victory in their opening three Group A matches.
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