<h3>Purpose/Objective(s)</h3> To assess the effect of radiotherapy (RT) on peripheral immune cells (B- and T-lymphocytes, Natural Killer (NK) cells) in prostate cancer (PCa) patients treated with different fractionation schedules. Here we report a preliminary analysis on the first 18 patients. <h3>Materials/Methods</h3> The present IRB approved prospective study was conducted at a single institution from February to October 2019; all patients referred for definitive, adjuvant or salvage radiotherapy for PCa were offered participation. Accrued patients were treated according to institutional guidelines/ongoing research protocols as it follows: stereotactic body radiation therapy (40 Gy/3 fractions; Group 1); definitive moderately hypofractionated RT (62 Gy/ 20 fractions; Group 2); postoperative moderately hypofractionated fractionated RT (66-69 Gy/30 fractions; Group 3). Target volumes were: prostate only (Group 1); prostate and seminal vesicles (Group 2); prostatic bed + pelvic lymph nodes (Group 3). All accrued patients underwent blood collection at baseline (PRE), 3 hours after the first dose of RT (3H) and at the end of RT (LAST). The immuno cell profiles were analyzed in isolated peripheral blood mononuclear cells, by multicolor flow cytometry, at each time point in terms of CD3, CD8, CD4, CD8/CD4-ratio, CD19 and CD3-, CD56+ expressions. Data were analyzed with the Wilcoxon-Mann-Whitney test. Statistical significance was claimed for <i>P</i> values < 0.05. <h3>Results</h3> 18 patients were accrued (5 pts Group 1; 6 pts Group 2; 7 pts Group 3). Baseline immunoprofiling did not demonstrate any significant difference among groups. Compared to PRE, both CD3 and CD4 cells increased significantly (<i>P</i> < 0.05) at 3H in all groups. CD8 cells decreased significantly at both 3H and treatment end in Group 1 (<i>P</i> = 0.043), but no statistically significant differences were noticed in Groups 2 and 3. The CD8/CD4-ratio decreased significantly in Group 1 at 3H (<i>P</i> = 0.04), while decreased after the last dose in both Groups 2 and 3 (<i>P</i> = 0.06). Compared to baseline, total NK cells decreased at 3H (Groups 1-2, <i>P</i> = 0.043; Group 3, <i>P</i> = 0.028), while increased for all 3 Groups at LAST. Finally, B cells were found to be decreased for both Groups 2 (<i>P</i> = 0.028) and 3 (<i>P</i> = 0.018) at LAST. <h3>Conclusion</h3> Systematic immunomonitoring at precise time points for PCa patients treated with different schedules/volumes shows different effects on lymphocyte subpopulations.