Performing a penalty kick in an international soccer tournament penalty shootout is an extremely stressful, emotional event (Jordet & Elferink-Gemser, 2012), often leading to the phenomenon of choking under pressure. Despite this, there has been an absence of scientific research on the development and effect of a high-pressure penalty kick protocol in professional football, which has also led to lack of research on apposite interventions under such pressure circumstances. To address this, Study 1 examined the effect of a high-pressure protocol on penalty shooting performance, psychological and psychophysiological response. The extent to which professional football players reported effects that are consistent with known mechanisms (distraction and self-focus) articulated in current theories of choking were examined. Findings indicated pressure, cognitive anxiety, respiration rate and bivariate variable error performance significantly increased (i.e., players were more variable in their grouping of shots) under pressure. Qualitative data indicated the high-pressure protocol predominantly evoked the distraction mechanism. Based on extant research in clinical psychology and resultant findings from Study 1, Study 2 examined whether a breathing intervention could be of potential benefit in mitigating distraction. Eighty-one male and female athletes undertook diaphragmatic pursed-lips breathing (PLB) training. Respiratory rate (RR) was examined across baseline, diaphragmatic-PLB without biofeedback, diaphragmatic-PLB visual biofeedback and end baseline conditions. Using a subsample of male professional footballers, Study 2 further examined the effect of diaphragmatic-PLB on psychological and psychophysiological response, and real-world application. Findings indicated the use of diaphragmatic-PLB resulted in decreased RR and perceptions of improved attentional/emotional control. Collectively, these mixed-method studies provided first insight into the experiences of professional footballers to a simulated high-pressure penalty kick protocol, the underlying mechanisms and the potential benefit of diaphragmatic-PLB in reducing distraction more generally. Future research is warranted to determine the benefit of diaphragmatic-PLB in improving emotional/attentional control across the dynamic penalty process.