Emotions are a core factor of learning. Studies have shown that multiple emotions are co-experienced during learning and have a significant impact on learning outcomes. The present study investigated the importance of multiple, co-occurring emotions during learning about human biology with MetaTutor, a hypermedia-based tutoring system. Person-centered as well as variable-centered approaches of cluster analyses were used to identify emotion clusters. The person-centered clustering analyses indicated three emotion profiles: a positive, negative and neutral profile. Students with a negative profile learned less than those with other profiles and also reported less usage of emotion regulation strategies. Emotion patterns identified through spectral co-clustering confirmed these results. Throughout the learning activity, emotions built a stable correlational structure of a positive, a negative, a neutral and a boredom emotion pattern. Positive emotion pattern scores before the learning activity and negative emotion pattern scores during the learning activity predicted learning, but not consistently. These results reveal the importance of negative emotions during learning with MetaTutor. Potential moderating factors and implications for the design and development of educational interventions that target emotions and emotion regulation with digital learning environments are discussed.