Transportation is a complex stressor made up of factors including weaning itself and withdrawal from feed and water. Therefore, transportation has the potential to negatively impact the health and welfare of weaned pigs. Pigs were transported for 32 h and measures of performance, physiology, and behavior were taken to assess piglet welfare. Treatment groups included pigs not weaned or transported (CON), weaned pigs provided with feed and water (WEAN+), weaned pigs not provided with feed and water (WEAN−), weaned and transported pigs provided with feed and water (TRANS+), and weaned and transported pigs not provided with feed and water (TRANS−). Body weight loss was different among treatments (p < 0.01). CON pigs had a 6.5% ± 0.45% gain in body weight after 32 h. WEAN+, WEAN−, TRANS+, and TRANS− groups all had a loss in body weight of 5.9% ± 0.45%, 7.8% ± 0.45%, 6.5% ± 0.45% and 9.1% ± 0.46%, respectively. The N:L was greater in all weaned pigs at 8 h compared to CON pigs (p < 0.01). WEAN− and transported pigs had significantly higher N:L than CON pigs from 8 h through 16 h, however, all treatment groups were similar to CON pigs after 16 h irrespective of provision of feed and water. Blood glucose levels were lower in transported and/or weaned pigs than CON pigs after 16 h irrespective of the provision of feed and water. TRANS+ females had higher creatine kinase (CK) levels than males (p < 0.05). After a 16 h transport period, TRANS− pigs had higher total plasma protein (TP) levels than all other treatment groups (p < 0.05). Significant changes in behavior were observed during and after transportation, which could also be indicative of stress. Overall, transportation and weaning had a negative effect on performance, physiology and behavior (both during and post-weaning) of pigs, especially when feed and water was not provided. Transporting pigs without feed and water for more than 24 h was a welfare concern as indicated by changes in body weight and physiology measures of stress.