Working on a moving platform can significantly impede human performance. Previous studies on moving vehicles have often focused on the overall impact on general task performance, whereas our study’s emphasis is on precise hand movements, exploring the interaction between body motion and the escalation of task difficulty. We recruited 28 participants to engage in reciprocal aiming tasks, following Paul Fitts’s setting, under both in-motion and stationary conditions. The task index of difficulty (ID) was manipulated by varying the width of the targets and the distance between the targets. We measured participants’ movement time (MT), performance errors, and monitored their eye movements using an eye-tracking device, heart rate (HR), and respiration rate (RR) during the tasks. The measured parameters were compared across two experimental conditions and three ID levels. Compared to the stationary conditions, the in-motion conditions degraded human aiming performance, resulting in significantly prolonged MT, increased errors, and longer durations of eye fixations and saccades. Furthermore, HR and RR increased under the in-motion conditions. Linear relationships between MT and ID exhibited steeper slopes under the in-motion conditions compared to the stationary conditions. This study builds a foundation for us to explore the control mechanisms of individuals working in dynamic and demanding environments, such as pilots in airplanes and paramedics in ambulances.