Research on the acute physiological response to a padel match is limited. The present study aimed to: (a) evaluate neuromuscular, urinary, and hematological responses after simulated padel competition (SC) and (b) analyze possible gender differences. In this study, 28 high-level padel players participated (men = 13, age = 26.83 ± 6.57 years; women = 15, age = 30.07 ± 4.36 years). The following parameters were analyzed before and after SC: neuromuscular (hand grip strength, squat jump (SJ), countermovement jump (CMJ), and Abalakov jump (ABK)), hematological (red blood cells, hemoglobin, and hematocrit), and urinary (pH, specific gravity, microalbuminuria, and red blood cells). Significant gender differences were found in neuromuscular and hematological responses, with men obtaining higher values (p < 0.05). For the SC influence, changes were noted in ABK and microalbuminuria (p < 0.05). The percentages of change in hand grip strength, SJ (height and watts), CMJ (height), and ABK (height) were higher for men than women (p < 0.05). SC negatively influenced the neuromuscular parameters to a greater extent in women. Our results could be related to gender differences in game actions, the temporal structure, and anthropometric and physiological characteristics. Game dynamics and a different organic response between male and female padel playing were confirmed.