Objectives To determine the extent of reproducibility of five facial expressions. Design Thirty healthy Caucasian volunteers (15 males, 15 females) aged 21 to 30 years had 20 landmarks highlighted on the face with a fine eyeliner pencil. Subjects were asked to perform a sequence of five facial expressions that were captured by a three-dimensional camera system. Each expression was repeated after 15 minutes to investigate intrasession expression reproducibility. To investigate intersession expression reproducibility, each subject returned 2 weeks after the first session. A single operator identified 3-dimensional coordinate values of each landmark. A partial ordinary procrustes analysis was used to adjust for differences in head posture between similar expressions. Statistical analysis was undertaken using analysis of variance (linear mixed effects model). Results Intrasession expression reproducibility was least between cheek puffs (1.12 mm) and greatest between rest positions (0.74 mm). The reproducibility of individual landmarks was expression specific. Except for the lip purse, the reproducibility of facial expressions was not statistically different within each of the two sessions. Rest position was most reproducible, followed by lip purse, maximal smile, natural smile, and cheek puff. Subjects did not perform expressions with the same degree of symmetry on each occasion. Female subjects demonstrated significantly better reproducibility with regard to the maximal smile than males (p = .036). Conclusions Under standardized conditions, intrasession expression reproducibility was high. Variation in expression reproducibility between sessions was minimal. The extent of reproducibility is expression specific. Differences in expression reproducibility exist between males and females.