(A) Example of bilateral eye movements in one mouse when it was head-fixed (top) compared to freely moving (bottom). Light purple, body still; dark purple, body actively moving, as measured using overhead video images (see Materials and methods). (B) Scatterplot of left and right eye positions in the example mouse from (A), during head fixation (black, left) and during free motion (purple, right). (C) Variability of eye position in head-fixed and freely moving mice. The standard deviation of eye position increased in head-free (dark purple) compared with head-fixed (black) mice (p=1.9 × 10−4, paired t-test, n = 7). Eye movements recorded in head-free mice were further subdivided into periods when the body was still (light purple) versus actively moving (medium purple) (p=0.146, paired t-test, n = 7). Grey lines, individual mice; black lines, example mouse in (A). (D) Scatterplots of velocity of the left and right eye during head fixation and free motion, for the example mouse in (A). (E) Mean eye speed was higher in freely moving than head-fixed mice (p=9.7 × 10−5, paired t-test, n = 7). In unrestrained mice, eye speed was highest during periods of active body movement compared to periods when the body was relatively still (p=3.6 × 10−4, paired t-test, n = 7). (F) Correlation between horizontal eye velocity and head velocity about the yaw axis in freely moving mice (n = 4). (G) Binocular coordination in head-fixed and freely moving mice. Left, mean eye divergence (horizontal angle between the two eyes) increased during free motion compared to head fixation (p=0.029, paired t-test, n = 7). Right, the standard deviation of eye divergence also increased during free motion (p=0.009, paired t-test, n = 7).