The German team of Annika Bruhn (23), Anna Dietterle (19), Helen Scholtissek (19) and Paulina Schmiedel (23), failed to swim the qualifying time for the Olympics at last August's world championships in Kazan.
The quartet then failed to swim the necessary standard times at last weekend's national trials in Berlin.
They will now not be allowed a final opportunity to make the Olympic mark at the European championships in London, from May 9-22, with head coach Henning Lambertz insisting: "they have already had enough chances".
The last time Germany won an Olympic gold in the event was 20 years ago in Atlanta and Australia are the reigning world and Olympic champions.
