Back in March, Louis laughed off the gay rumours, telling radio station KXXAS that their US fans "genuinely, seriously think that we’re in a relationship. It’s so funny!"
He also revealed that some devotees were so convinced that they were an item, that they had nicknamed the lads Larry Stylinson.
However, it seems the joke has gone too far for Louis, with the singer insisting in an interview with Storyboard that he only has eyes for girlfriend, Eleanor Calder.
Tomlinson stated: "This is a subject that was funny at first but now is actually hard to deal with, as I am in a relationship.
"Me and Harry are best friends. People look into our every move - it is actually affecting the way me and Harry are in public."
He added: "We want to joke around, but there seems to be a different rumour every time we do anything; I act the same way with Harry as I do any of the other boys."
Watch MTV News on the hour every hour on MTV - Sky Channel 126 and Virgin Channel 134