Influenza A viruses circulating in humans from ～1950 to ～1987 featured a non-structural (NS1) protein with a C-terminal extension of seven amino acids. The biological significance of this NS1 elongation remained elusive. We observed that replication kinetics of wild-type A/Hong Kong/01/68 (H3N2) and a mutant encoding a truncated NS1 were indistinguishable in most experimental systems. However, wild-type virus out-competed the mutant during mixed infections, suggesting that the NS1 extension conferred minor growth advantages.