The objective of this study was to evaluate the effects of temperature (
T
)/relative humidity (
RH
) combinations and exposure duration (
D
) on the muscle tissue characteristics of layer pullets during simulated transport. While layer pullets are not processed for meat, muscle physiology can be used as an indicator to assess welfare. Pullets (n = 240) were randomly assigned to 1 of 5 T/RH combinations (−15°C uncontrolled RH [−15], 21°C 30%RH [21/30], 21°C 80%RH [21/80], 30°C 30%RH [30/30], and 30°C 80%RH [30/80]) and 2 D (4 or 8 h) in a 5 x 2 factorial arrangement (3 replications). Birds were weighed before exposure, crated (density 45.5 kg/m
2
) and exposed to the conditions above. After exposure, birds were weighed (live shrink calculated) and slaughtered using a small-scale facility. Postslaughter, carcasses were eviscerated, and an initial pH was obtained from the right breast and thigh. Final breast and thigh pH and color values (lightness [L∗], yellowness [b∗], and redness [a∗]) were obtained 30 h postslaughter. Left breast muscles were frozen and analyzed for thaw and cook loss 4 wk postslaughter. Data were analyzed as a randomized complete block design via ANOVA (Proc Mixed; SAS 9.4), with farm of origin as block. Differences were considered significant when
P
≤ 0.05. Live shrink (kg) was higher for pullets exposed to 30/30 and 30/80 compared with those exposed to 21/80 (
P
= 0.04) and for pullets exposed for 8 h compared with 4 h (
P
< 0.01). Breast muscle thaw loss (%) was higher in pullets exposed for 4 h compared with 8 h (
P
= 0.01). Breast and thigh muscle a∗ were higher for pullets exposed to 30/30 compared with 21/30 (
P
= 0.02). Thigh muscle b∗ was lower for pullets exposed to -15 compared with 21/80 (
P
= 0.05). Breast b∗ was higher for pullets exposed for 8 h compared with 4 h (
P
= 0.04). The results from this study demonstrates that increasing exposure D had minor effects on pullet muscle characteristics. In addition, layer pullets coped well with thermal stressors associated with simulated transport.
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