Mastitis and associated pain have been identified as a major health and welfare problem affecting dairy cows, however little is known about how cows emotionally experience this illness. Qualitative behaviour assessment (QBA) is a 'whole animal' methodology for assessing animal emotion, through description and quantification of the expressive qualities of an animal's dynamic style of behaving (eg as relaxed, anxious). The aim of this study was to use QBA to investigate whether emotional expression in dairy cows is affected by an experimental intra" mammary challenge (mastitis) with Escherichia coli, and to investigate the relationship of QBA scores with nine other clinical, physiological and behavioural welfare indicators. Six Holstein-Friesian cows were inoculated with E. coli in one healthy quarter. Evolution of the disease was assessed using bacteriological growth and somatic cell counts (SCC). The cows' response to the challenge was assessed using QBA, clinical observations, data loggers, rumen temperature sensor, and physiological indicators (inflammation, stress) at ten time-points defining the phase of the disease: before inoculation (Phase 0: 0 h), in the pre-clinical Phase (Phase 1: 8 h), in the acute phase (Phase 2: 12 h, 16 h, 24 h) and in the remission phase (Phase 3 : 32 h, 40 h, 56 h, 64 h and 80 h post-inoculation (hpi)). Principal Component Analysis of QBA scores identified two main dimensions of cow expression: PC1, ranging from active/vigorous/happy/bright to suffering/dejected/lethargic, and PC2, ranging from fearful/tense/anxious to confident/calm/relaxed, together explaining 58% of the total variation. QBA PC1 and PC2 scores varied with mastitis phases: QBA PC1 scores decreased by 4.09 in Phase 2, and by 1.98 in Phase 3, reflecting suffering/dejected/lethargic expressivity. QBA PC2 scores decreased by 1.91 in Phase 3, reflecting a confident/calm/relaxed expressivity. Clinical and physiological welfare indicators were associated with QBA. The higher the udder severity score, the body temperature, the concentrations of cortisol, SAA, TNF-alpha, and IL-1 beta, the more the cows were suffering/dejected/lethargic (PC1) (coefficients:-0.51,-0.92,-2.46, 7.52 x 10-5,-0.72,-1.13 respectively). These findings indicate that dairy cows experienced negative emotional state in the acute phase and positive emotional state in the remission phase of mastitis. This suggests that provision of pain-relief treatment during mastitis may improve animal welfare, and potentially lead to faster disease remission. However the sample size of this study was small, and larger controlled studies are needed to further investigate these findings and hypotheses. The sensitivity of QBA in this small study suggests it could potentially be a useful tool for E. coli mastitis detection.