Background: The main characteristics of CrossFit® are that it is constantly varied and of high-intensity. The planning of CrossFit® training sessions can be differentiated by modality, frequency, rest intervals, variation, and duration, which can elicit different responses. Purpose: The aim of the present study was to compare the acute effects of a short-duration (Fran) and long-duration (Cindy) session of CrossFit® on tissue damage (creatine kinase-CK), autonomic responses, and fatigue in experienced CrossFit® practitioners. Methods: Thirteen men (31.2 ± 3.2 years) with 5.72 ± 2 years of experience participated in a crossover study with a counterbalanced design, where they completed both the short-duration and long-duration CrossFit® sessions. Blood samples were collected to check tissue damage via CK, autonomic responses were verified through heart rate variability (HRV), and the fatigue by countermovement jump (CMJ) at the following moments: pre-workout, immediately after (IA), 24- and 48-hour after the training session. Results: A statistically significant increase in CK was observed for Fran at 24 hours versus pre-workout (p = .029), while for Cindy there was a statistically significant increase in CK IA (p = .002) and 24 hours versus at pre-workout (p = .002). HRV showed lower values (p < .05) IA for the time domain in Fran and for the frequency domain in both training sessions. HRV returned to pre-workout values at 24 hours. CMJ did not show significant differences between moments and training sessions. The present study showed that CK significantly increased from baseline just after the long-duration session and was increased 24 hours after both. HRV significantly decreased IA, except in the frequency domain for the shortest training session (Fran). CMJ did not change through 48 hours after training, regardless of the training session. Conclusions: Overall, these findings emphasize the importance of considering both the intensity and duration of CrossFit® workouts when assessing their impact on physiological responses and potential risks.