Autoimmune and other chronic inflammatory diseases (AID) are prevalent diseases which can severely impact the quality of life of those that suffer from the disease. In most cases, the etiology of these conditions have remained unclear. Immune responses that take place e.g. during natural infection or after vaccination are often linked with the development or exacerbation of AID. It is highly debated if vaccines induce or aggravate AID and in particular adjuvants are mentioned as potential cause. Since vaccines are given on a large scale to healthy individuals but also to elderly and immunocompromised individuals, more research is warranted. Non-specific induction of naïve or memory autoreactive T cells via bystander activation is one of the proposed mechanisms of how vaccination might be involved in AID. During bystander activation, T cells unrelated to the antigen presented can be activated without (strong) T cell receptor (TCR) ligation, but via signals derived from the ongoing response directed against the vaccine-antigen or adjuvant at hand. In this study we have set up a TCR transgenic T cell transfer mouse model by which we were able to measure local bystander activation of transferred and labeled CD4⁺ T cells. Intramuscular injection with the highly immunogenic Complete Freund’s Adjuvant (CFA) led to local in vivo proliferation and activation of intravenously transferred CD4⁺ T cells in the iliac lymph node. This local bystander activation was also observed after CFA prime and Incomplete Freund’s Adjuvant (IFA) boost injection. Furthermore, we showed that an antigen specific response is sufficient for the induction of a bystander activation response and the general, immune stimulating effect of CFA or IFA does not appear to increase this effect. In other words, no evidence was obtained that adjuvation of antigen specific responses is essential for bystander activation.