Prolonged driving could induce neuromuscular fatigue and discomfort since drivers have little opportunity to adjust their position. However, better car seat design could play a major role in limiting these effects. This study compared the effect of two different seats (S - soft and F - firm) on neuromuscular fatigue and driver's perceived discomfort during prolonged driving, also assessing the effect of different road types on neuromuscular activity. Twenty participants performed two 3-h driving sessions, one for each seat, on a static simulator. Every 20 min, participants self-evaluated their level of whole-body and individual body-area discomfort. Surface electromyography (sEMG) was recorded for eight muscles including Trapezius descendens (TD), Erector spinae longissimus (ESL), Multifidus (MF), Vastus lateralis (VL) and Tibialis anterior (TA) throughout the driving sessions. Moreover, an endurance static test (EST) was performed prior to and after each driving session. Whole-body discomfort increased with time with both seats, but no difference in discomfort scores was observed between seats throughout the driving sessions. The highest discomfort scores were for neck and lower back areas with both seats. Neuromuscular fatigue was revealed by a shorter endurance time in post-driving EST for both seats. EMG recordings showed different neuromuscular fatigue profiles for the two seats, with earlier onset of fatigue for S. Despite the lack of difference in perceived discomfort level, the two seats have different impacts: the softness of S induces greater activity of the lower back muscles, while F offers greater support for the lower back.