Table 2 Effects of handling method and hold duration on anxiety responses in an open field test.

	Time in central zone (s)		Visits to central zone (n)		Time per central zone visit (s)		Latency to arena edge (s)		Stretched attend (n)
Handling method	F_2,54 = 15.62	P < 0.0001	F_2,54 = 4.54	P = 0.015	F_2,54 = 22.88	P < 0.0001	F_2,54 = 25.01	P < 0.0001	F_2,54 = 0.35	P = 0.71
Handling duration	F_3,54 = 2.14	P = 0.11	F_3,54 = 3.29	P = 0.027	F_3,54 = 0.34	P = 0.80	F_3,54 = 1.14	P = 0.34	F_3,54 = 2.00	P = 0.13
Sex	F_1,54 = 15.85	P = 0.0002	F_1,54 = 20.50	P < 0.0001	F_1,54 = 0.47	P = 0.50	F_1,54 = 1.21	P = 0.28	F_1,54 = 0.002	P = 0.96
Method × duration	F_6,54 = 0.93	P = 0.49	F_6,54 = 0.64	P = 0.70	F_6,54 = 0.68	P = 0.67	F_6,54 = 0.54	P = 0.84	F_6,54 = 1.54	P = 0.18
Method × sex	F_2,54 = 0.44	P = 0.65	F_2,54 = 0.37	P = 0.69	F_2,54 = 0.25	P = 0.78	F_2,54 = 0.24	P = 0.79	F_2,54 = 0.97	P = 0.38
Duration × sex	F_3,54 = 1.39	P = 0.26	F_3,54 = 0.68	P = 0.57	F_3,54 = 1.38	P = 0.26	F_3,54 = 0.51	P = 0.68	F_3,54 = 0.43	P = 0.74
3 way interaction	F_6,54 = 0.78	P = 0.59	F_6,54 = 1.72	P = 0.13	F_6,54 = 0.85	P = 0.54	F_6,54 = 0.81	P = 0.57	F_6,54 = 1.20	P = 0.32
Planned contrasts:
tail vs tunnel		P < 0.0001		P = 0.18		P < 0.0001		P < 0.0001		P = 0.85
tail vs cup		P = 0.18		P = 0.005		P = 0.038		P = 0.14		P = 0.47

Mice had previously been picked up by their assigned method and held for different durations (2, 10, 30 or 60 s) for 5 daily handling sessions. Mice were delivered to the centre of the open field arena using their assigned method (data shown in Fig. 1C–E and Supplementary File 1). Repeated measures ANOVAs, taking into account two mice per cage as a within-subjects factor, with planned contrasts between tail and non-aversive tunnel or cup methods (time in central zone and time per visit log transformed to meet assumption of parametric analysis). Significant effects (P < 0.05) are in bold.

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