Is there any Performance benefit in Nodejs using singleton and non-singleton pattern ?

I am working on an API using express and was wondering if there is any performance overhead of abstracting my callbacks of express routes to a separate JS file . The thought which is leading to confusion is the fact "that every invocation has some resource cost attached to it"

Is there any performance advantage if I use the inline callback instead of a callback function defined in a separate JS file exposed via module.export.

via Harshwardhan