Mulerius was born Nicolaas des Mulier(s), son of Pierre des Muliers and Claudia Le Vettre. He grew up in Bruges, where he was taught by Cruquius, among others. Mulerius first studied Philology, Philosophy and Theology and from 1582 he also studied Medicine and Mathematics at the University of Leiden, where Lipsius, Vulcanius, Snellius and Heurnius were teachers. In 1589 he married Christina Six and set up practice for 13 years in Harlingen. In 1603 he became leading physician in Groningen, in 1608 he took the position as school master of the Leeuwardengymnasium. From 1614 he was professor in medicine and mathematics at the Groningen University. From 1619 – 1621 and 1626 – 1630, he was in charge of the library of the University of Groningen.
An account of the life of Ubbo Emmius, written by Nicolaus Mulerius, was published, with the lives of other professors of Groningen, at Groningen in 1638, eight years after Mulerius' death.
Mulerius married Christina Maria Six (1566-1645) in 1589 in Amsterdam. Among their children were Petrus Mulerius (pt) (1599-1647), who would become professor at Groningen in physics and botany from 1628, and Carolus Mulerius (1601-1638) who wrote the first grammar of Spanish in Dutch.
^Studying in Groningen Through the Ages: A History of the University of Groningen and the First History of the Department of English in the Netherlands. Groningen: Groningen University Press, 2014, p. 33. ISBN 978-90-367-7125-2
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