Abstract Krzysztofik, M, Spieszny, M, Trybulski, R, Wilk, M, Pisz, A, Kolinger, D, Filip-Stachnik, A, and Stastny, P. Acute effects of isometric conditioning activity on the viscoelastic properties of muscles and sprint and jumping performance in handball players. J Strength Cond Res 37(7): 1486–1494, 2023—The effects of conditioning activity (CA) on muscle stiffness are currently unknown, suggesting that maximum CA effort can increase or decrease the stiffness of involved muscle groups. Therefore, this study aimed to investigate the effect of maximal isometric half-squats on the viscoelastic properties of muscles and postactivation performance enhancement (PAPE) in sprints and jumps. Twelve handball players underwent a standard warm-up and baseline assessment of muscle stiffness and tone of vastus lateralis and gastrocnemius medialis muscle, followed by 20-m sprint with intermediate measures at 5 and 10 m and countermovement jump. The PAPE was assessed by repeating the tests (at 4th, 8th, and 12th minute post-CA) after a CA protocol consisting of 3 sets of 3-second maximal isometric half-squats (EXP) or a control condition (CTRL) without any CA. The vastus lateralis stiffness in the 4th and 12th minute and muscle tone in the 4th minute post-CA significantly decreased compared with baseline (p = 0.041, ES = 0.57; p = 0.013, ES = 0.52; p = 0.004, ES = 0.81, respectively) in the EXP condition. The 20-m sprint time significantly decreased at all post-CA time points compared with the baseline for the EXP condition (p < 0.033) and the after values in the CTRL condition (p < 0.036). In comparison to baseline, the 10-m sprint time decreased in the eighth minute post-CA (p = 0.021; ES = 0.82) in the EXP condition. Moreover, it was significantly lower at the 8th and 12th minute post-CA (p = 0.038; ES = 0.71 and p = 0.005; ES = 1.26) compared with that time points in the CTRL condition. The maximal isometric half-squats effectively improved sprint performance and significantly decreased vastus lateralis tone and stiffness. These findings offer new insights into the assessment of viscoelastic properties for evaluating the fatigue or potentiation state, which requires further investigation.