Both sets of players had already left the pitch for the interval when the technology flagged up a handball at the end of the first period, reports The Guardian football journalist Andy Brassell on social media.
The two teams were ordered to return to the field of play so Pablo de Blasis could take the penalty, which he buried to give the home side the lead.
Now this is special, even for VAR. Half-time whistle goes in Mainz v Freiburg, players go off, then pen to Mainz given for handball on last action of half. Players called back out, De Blasis scores, players go off again.— Andy Brassell (@andybrassell) April 16, 2018
De Blasis later doubled the home side's lead in the 79th minute and the game finshed 2-0.
The VAR incident is bound to spark discussion about the pros and cons of the technology, which has divided opinion during trials ahead of its use at this summer's World Cup finals.