Penalty shootouts (PSO) in international elite football tournaments are of great importance for team success. We aimed to investigate appearance and success rates in PSO of starters vs substitute players in male and female elite football tournaments before and after the introduction of the 5+1 substitution rule in 2020. We analysed 428 penalty kicks in 43 PSO from 2013–2025 in FIFA World Cups (Men: 13/Women: 5), UEFA Euros (Men: 10/Women: 7) and Olympic Football Tournaments (Women: 8). In male tournaments, the fraction of substitute players involved in PSO increased post-2020 compared to pre-2020 (61.2 vs. 28.6%, p < .001), but not in female tournaments (37.0 vs. 25.6%, p=.20). Exploratory simple-effect analyses showed that the predicted conversion rate among starters was lower post-2020 than pre-2020 (OR=0.54, 61.9 vs 75.1%, p = .029), whereas no significant change was observed among substitutes (OR=0.80; 63.2 vs. 57.8% p = .510). The overall scoring rates in the male and female tournaments, respectively, were 71.4 and 74.4% before and 67.0 and 61.4% after the implementation of the 5+1 substitution rule. The present study suggests an impact of the 5+1 substitution rule on appearance of substitute players in penalty shootouts in international tournaments, especially for males, and a reduction in the scoring rates for starters. This development is of value to consider for coaches when preparing for the knockout games in international tournaments.