Introduction: Intensified training and sufficient recovery is required to improve athletic performance. Heavy training has been reported as immunosuppressive in athletes, possibly due to reduced immunosurveillance with increased exercise stress. A maladaptation of the hypothalamic pituitary adrenal (HPA) axis has been shown after periods of intensified training; specifically, a blunted cortisol response to an exercise stress test has been reported. Activation of the HPA axis elevates dendritic cell (DCs) numbers. DCs are key antigen presenting cells that present antigen to T cells to initiate the required immune response. The link between training stress, the HPA axis and DCs has yet to be examined. Before investigating whether DCs become dysfunctional with intensified training, a reproducible response of these cells to an exercise stress test must be confirmed. Reproducible cortisol responses to alternating blocks of 1-minute cycling at 55% maximum power output (W_max) and 4-minutes cycling at 80%W_max for 30-minutes (55/80) have been shown, and 20-minutes cycling at 80%VO_2max can elevate total DCs numbers. The 55/80 is based on percentage of work rate maximum, but large differences in homeostatic perturbations i.e. O₂ uptake kinetics and blood lactate responses are likely between participants in the 55/80. Therefore, submaximal anchors such as the ventilatory threshold (VT₁) should be used to prescribe intensity. Aim: Therefore, this study aims to assess the reproducibility of the DC and T cell responses to an adapted version of the 55/80 utilising VT₁ to prescribe intensity. Methods: 12 healthy males (age, 26.4 ± 5.8 years; VO_2peak, 48.58 ± 7.14 ml/kg/min) cycled for 1-minute at a work rate 20% below VT₁ and 4-minutes at 50% between VT₁ and VO_2max for 30-minutes (20/50 exercise test) with blood samples pre, post and 30-minutes post. This was repeated on two occasions, 2-7 days apart and at the same time of day. Using flow cytometry, total DCs were defined at Lineage– (CD3, CD19, CD20, CD14, CD56) HLA-DR+ and subsequently identified as plasmacytoid (CD11c- CD123+) (pDCs) or myeloid (CD11c+ CD123-) (mDCs). T-helper cells were identified as being CD3+CD4+ and T-cytotoxic cells were identified as being CD3+CD8+. Two-way repeated measures ANOVAs were used for all variables apart from pDCs which were analysed via a Wilcoxon signed-rank test for main effects of trial and post-hoc time effects, and a Friedmans test for main effect of time. Results: No significant effect of trial (P> 0.05) or interaction effects (P> 0.05) were found for any variable. A significant main effect of time for all variables were found with immune cell counts increasing from pre- to post-exercise and decreasing to baseline 30-minutes post-exercise (P< 0.001), apart from pDCs which remained elevated 30-minutes after exercise. Intraclass correlation coefficients showed excellent reliability for CD3+ T cells, CD8+ T cells and pDC responses between trials (ICC >0.75) and good reliability for Total DCs, mDCs and CD4+ T cells (ICC 0.6-0.74). Conclusions: These results suggest that the 20/50 exercise test induces reproducible DC and T-cell count changes, which, implemented before and after a period of intensified training, may highlight the negative states of overtraining.