Bus drivers often work irregular hours or split shifts and their work involves high levels of stress. These factors can lead to severe sleepiness and dangerous driving. This study examined how split shift working affects sleepiness and performance during afternoon driving. An experiment was conducted on a real road with a specially equipped regular bus driven by professional bus drivers. The study had a within-subject design and involved 18 professional bus drivers (9 males and 9 females) who drove on two afternoons; one on a day in which they had driven early in the morning (split shift situation) and one on a day when they had been off duty until the test (afternoon shift situation). The hypothesis tested was that split shifts contribute to sleepiness during afternoon, which can increase the safety risks. The overall results supported this hypothesis. In total, five of the 18 drivers reached levels of severe sleepiness (Karolinska Sleepiness Scale ≥8) with an average increase in KSS of 1.94 when driving in the afternoon after working a morning shift compared with being off duty in the morning. This increase corresponded to differences observed between shift workers starting and ending a night shift. The Psychomotor Vigilance Task showed significantly increased response time with split shift working (afternoon: 0.337s; split shift 0.347s), as did the EEG-based Karolinska Drowsiness Score mean/max. Blink duration also increased, although the difference was not significant. One driver fell asleep during the drive. In addition, 12 of the 18 bus drivers reported that in their daily work they have to fight to stay awake while driving at least 2-4 times per month. While there were strong individual differences, the study clearly showed that shift-working bus drivers struggle to stay awake and thus countermeasures are needed in order to guarantee safe driving with split shift schedules.