Background <p>The study aimed to assess the acute effects of overcoming isometric split squats on subsequent single-leg drop jump (DJ) performance and the contralateral effect in volleyball players, considering training status differences.</p> Methods <p>Ten male elite (ELI) and ten amateur (AMA) volleyball players took part in two experimental sessions in which they performed conditioning activity (CA) consisted of 3 sets of overcoming isometric 3-second split squats, differing in the limb used: dominant (DL) or non-dominant (ND-L). Single-leg DJ was performed before and at the 4th and 8th minute post-CA for both limbs. The best post-CA attempt in terms of jump height (JH) was analyzed.</p> Results <p>Results revealed a significant increase in jump height (JH) in the non-dominant limb (ND-L) from 13.7 ± 2.6&#xa0;cm to 15.5 ± 2.7&#xa0;cm after CA performed by the ND-L (<i>p</i> = 0.001; Hedge’s g = 0.65). Similarly, the reactive strength index (RSI) of the ND-L improved from 0.40 ± 0.06 to 0.45 ± 0.08 (<i>p</i> = 0.008; g = 0.58). Additionally, contact time in the ND-L increased significantly from 342 ± 36 ms to 375 ± 42 ms after CA performed by the dominant limb (<i>p</i> = 0.001; g = 0.66). In the elite (ELI) group, JH significantly increased from 16.4 ± 2.4&#xa0;cm to 18.3 ± 3.3&#xa0;cm (<i>p</i> &lt; 0.001; g = 0.79), while RSI in the dominant limb (DL) improved from 0.47 ± 0.06 to 0.53 ± 0.07 (<i>p</i> = 0.011; g = 0.68).</p> Conclusions <p>Results suggest that the examined CA effectively induces the post-activation performance enhancement in DJ among elite volleyball players, with a predominantly local impact on the limb executing the CA.</p> Trial registration <p>NCT06459050 (Retrospectively registered).</p>