The Prisoner’s Dilemma is one of the most classic formats for exploring the principle of direct reciprocity. Although numerous theoretical and experimental studies have been conducted, little attention has been paid to the divergence between theoretical predictions and actual human behaviour. In addition, there are two additional essential challenges of experimental research. First, most experimental approaches have focused on games in which two players decide their actions simultaneously, but little is known about alternating games. Another is that there are few experiments on voluntary participation. Here, we conducted experiments on simultaneous games, alternating games, and games with and without voluntary participation for a total of four game patterns and examined the deviation from theoretical predictions for each. The results showed that, contrary to theoretical predictions, humans chose cooperation even after being exploited. We also observed that, with or without voluntary participation, people tended to take the same action they had taken in the previous round. Our results indicate that to understand the mechanisms of human behaviour, we need to integrate findings from behavioural science, psychology, and game theory.