Most healthy adults have long lasting influenza specific memory CD8 T cells in peripheral blood, primed by previous infection with influenza viruses. Relatively high frequencies of memory T cells with increased proliferative capacity form the basis for enhanced and accelerated protection upon re-infection. Memory cells are characterized by frequent expression of the IL-7 receptor CD127 and the costimulatory molecules CD28 and CD27. This is the case for the majority of influenza specific T cells in healthy adults, but other subsets of influenza specific T cells have different phenotypes and unknown functions. Here we show that up to ~20% of HLA-A*0201 / influenza matrix protein58-66 specific T cells from healthy donors did not express CD127 and/or CD28. In contrast to the majority of CD28pos cells, granzyme B and perforin were frequently expressed by CD28neg cells, suggesting that they may be effector cells. With a newly developed flow cytometry-based cytotoxicity assay we show that these cells are indeed capable to lyse target cells in an antigen specific manner, directly ex vivo without prior stimulation, demonstrating the existence of influenza specific effector T cells in healthy humans. Sequencing of TCR alpha- and beta-chains revealed that the response to this epitope was dominated by ~1-3 T cell clonotypes. Interestingly, identical clonotypes were found as memory and effector cells, demonstrating for the first time that T cell memory is maintained by clonotypes that persist simultaneously in multiple differentiation stages. The data suggest that efficient secondary (memory) responses against influenza virus may depend on both, rapid expansion of memory cells and immediate function by long term persisting cytotoxic effector cells. Finally, these results reveal a surprising capacity of the human immune system for long term maintenance of circulating effector cells in absence of antigen, a property that deserves further investigation since it may be exploited for novel immunotherapy of minimal residual diseases.