Background: Unsettled infant behaviour is a common problem of infancy without known aetiology or effective management, and it is costly in both social and economic terms. Some osteopaths propose that musculoskeletal dysfunction contributes to unsettled infant behaviour, yet reported improvement following osteopathic treatment is anecdotal. A primary issue is the absence of a measurement tool to test musculoskeletal dysfunction in infants. Aims and Objectives: This research aimed to investigate: the reliability and validity of the infantile postural asymmetry (IPA) measurement scale; whether there was a relationship between IPA and unsettled infant behaviour as measured by the Revised Infant Behavior Questionnaire – short form (IBQ-Rs); and whether any relationship between IPA and unsettled infant behaviour was mediated by, or confounded with, the demographic variables of age, sex, birth weight and weight gain in twelve- to sixteen-week-old infants. Methods: Fifty-eight infants aged twelve- to sixteen-weeks-old were recruited through public health clinics, and their behaviour was assessed using the parent-report IBQ-Rs. Infantile asymmetry was measured using observer ratings of spontaneous movements in the IPA scale. A quantitative cross-sectional observational design was used to investigate the relationship between IPA and unsettled behaviour. Results: An association between unsettled behaviour and musculoskeletal dysfunction was not found in twelve- to sixteen-week-old infants using the IPA measurement scale. Ratings for the trunk convexity parameter of the IPA scale were unreliable and excluded from statistical analysis. A significant difference between high and low cervical rotation deficit groups for Surgency was detected in female babies and needs further examination. Some subsets of the IBQ-Rs were unstable when measuring behaviour in twelve- to sixteen-week-old infants. Future research targeting infants younger than twelve-weeks-old, and presenting with unsettled behaviour, is indicated. Conclusion: A causal relationship between unsettled infant behaviour and musculoskeletal dysfunction is still unproven. The literature suggests benefits associated with a consistent approach to providing parents with information, support and advice on normal behaviour patterns and optimal handling of infants. Non-specific effects cannot be ruled out in reported improvements following osteopathic treatment. Management strategies require early implementation and a multidisciplinary approach. The absence of common terminology in infant behaviour problems is an obstacle in cross-professional communication. A role for osteopathy may be in developing a shared language to facilitate management and research, and to examine the importance of positioning and handling practices on infant asymmetry and the relationship with the musculoskeletal system.