We investigated the association of the number of eating occasions and energy intake with alertness and gastrointestinal (GI) complaints in nurses during their night shift. During this observational study we collected data on anthropometrics and demographics, eating frequency, energy intake, alertness and GI complaints in 118 healthy female nurses, aged 20 to 61 years. Nurses completed an alertness test (psychomotor vigilance task) during the night shift and a 24-hr dietary recall and a questionnaire about GI complaints after the night shift. This was repeated three times, always on the first night shift in a night shift series. The number of eating occasions during the night shift was negatively associated with reaction times (β = -4.81 ms, 95% confidence interval [CI] -9.14 to -0.48; p = .030), and number of lapses (β = -0.04, 95% CI -0.07 to -0.00; p = .030). However, the number of eating occasions was not associated with subjective alertness and GI complaints. Energy intake during the night shift was not associated with objective or subjective alertness or with GI complaints. These associations were independent of caffeine intake, age, body mass index and dependence among the repeated measurements. The present study showed that eating frequency was positively associated with objectively measured alertness levels in female nurses during the night shift. The results need to be confirmed in an intervention study, where also timing, size and composition of the meal will be taken into account. In practice, optimising nutritional guidelines on these aspects could lead to faster responses, less (medical) errors, and a better wellbeing of night shift workers.