Inhaled beta-2 adrenoceptor agonists (iβ2A) are routinely used as bronchodilators in the treatment of asthma. However, their cardiac effects in athletes are scarcely examined. Thus, the aim of this study was to evaluate the effects of iβ2A on left ventricular (LV) systolic function (SF) by echocardiography in healthy, non-asthmatic female and male endurance athletes. A randomized, double-blinded, placebo-controlled, balanced, 4-way complete block cross-over study was conducted. Twenty-four healthy athletes (12f/12m: 22.9 ± 2.7/24.4 ± 4.6 years) randomly completed 4 study arms (placebo; salbutamol; formoterol; formoterol + salbutamol). After inhalation of the study medication, the participants performed a 10-min time trial (TT) on a bicycle ergometer. After each TT an echocardiography was performed to determine LVSF. Blood samples were collected pre, post, 3 h and 24 h post TT. In females, total serum concentrations for salbutamol and formoterol were higher. LV ejection fraction (LVEF) and LV global longitudinal strain (LVendoGLS) showed a treatment effect for the whole study group (p < 0.0001) and a sex effect on LVEF (p = 0.0085). In women, there was a significant treatment effect for all medication arms (at least p ≤ 0.01) both on LVEF and LVendoGLS. In men only formoterol and formoterol + salbutamol displayed a treatment effect on LVEF (p = 0.0427, p = 0.0330; respectively), whereas on LVendoGLS only formoterol + salbutamol was significant (p = 0.0473). The iβ2A significantly influenced LVSF after an acute bout of exercise in healthy endurance athletes. These effects were even more pronounced when combining both iβ2A that supports a dose-dependent effect on cardiac function. Moreover, female athletes had higher serum concentrations of β2 agonists and stronger effects on LVSF compared to male athletes. This is mainly explained by differences in body weight and related plasma volume and may indicate a potential risk when increasing dose above the tested concentrations. Trial registration: At the European Union Drug Regulating Authorities Clinical Trials (Eudra CT) with the number 201,500,559,819 (registered prospectively on 09/12/2015) and at the German register for clinical studies (DRKS number 00010574 registered retrospectively on 16/11/2021).