Few studies have examined the impact of a preseason training intervention through systematic measures in Pro D2 rugby union (RU). Therefore, this study aimed to describe the effects of 12 weeks of preseason training (three blocks) on body composition, running performance, biochemical markers, and workload (WL) variation in professional RU players. Physiological (physical and biochemical) responses to preseason WL were analyzed by examining changes in anthropometric characteristics, Yo-Yo intermittent recovery level 1 (Yo-Yo IR1) test, blood samples (BS), Hooper index (1–7), the 10-Hz global positioning system (GPS), and session rating of perceived exertion (s-RPE) in nineteen elite male players. Changes throughout the preseason were analyzed using the one-way and mixed-model analysis of variance. Significant (p < 0.01) improvements occurred in anthropometry and Yo-Yo IR1 running performance in forwards and backs. Total distance (p < 0.01) and impact (p < 0.05) during the second block were meaningfully higher than the other two blocks, with backs showing higher values than forwards. As expected, WL decreased significantly (p < 0.01) during the last training block. The WL variations were correlated with changes in biochemical markers over the preseason period. The collected data can be used for i) profiling French Pro D2 rugby championships players, ii) establishing effective training strategies, and iii) setting preseason WL expectations.