The objective of this randomized controlled trial was to determine if providing a rest stop including provision of feed and water between periods of continuous transportation mitigates the effect of long-distance transportation on markers of energy status and hydration in calves transported by road for 16 h. This study was conducted between September 2022 to January 2023 and included male and female Holstein and crossbred dairy calves ≥7 d old from 2 commercial dairy farms in Ontario, Canada (n = 96). On the day before transportation, calves were enrolled and randomly assigned to one of 2 treatment groups: (1) continuous transportation by road for 16 h or (2) 8 h of transport, 8 h of rest, and a further 8 h of transport to a single calf-raising facility. Calves that received a rest stop were fed 2 L of milk replacer at the time of unloading for the rest period and again immediately before reloading for the second leg of the journey. Blood samples were collected before and after transportation as well as daily for 3 d following arrival to the calf-raising facility. Serum was analyzed for concentrations of BHB, nonesterified fatty acids (NEFA), haptoglobin, and creatine kinase, and acid-base disturbances were evaluated within 1 h of collection of whole blood to assess glucose, sodium, potassium, ionized calcium, hematocrit, hemoglobin, pH, base excess, partial pressure of oxygen, total carbon dioxide, bicarbonate, partial pressure of carbon dioxide, and oxygen saturation. Mixed linear regression models with repeated measures were built to assess the effect of treatment group, age at transportation, breed, and sex on these parameters. Immediately after transportation, calves that received the rest period had lower BHB (-68.04 μmol/L, 95% CI: -99.59 to -36.49), NEFA (-0.14 mmol/L, 95% CI -0.22 to -0.07), and sodium (-1.10 mmol/L, 95% CI = -2.18 to -0.02), and higher glucose (0.48 mmol/L, 95% CI = 0.21 to 0.74), potassium (0.27 mmol/L, 95% CI = 0.11 to 0.43), ionized calcium (0.06 mmol/L, 95% CI = 0.03 to 0.08), and oxygen saturation (8.76%, 95% CI = 1.61 to 15.91) compared with calves that were continuously transported for 16 h. Additionally, calves transported between 11 and 12 d old had lower hematocrit (-1.22%, 95% CI = -2.19 to -0.25), hemoglobin (-3.07 g/L, 95% CI = -5.70 to -0.43), haptoglobin (-0.13 g/L, 95% CI = -0.23 to -0.02), and potassium (-0.13 mmol/L, 95% CI = -0.22 to -0.04) and higher sodium (0.83 mmol/L, 95% CI = 0.03 to 1.63) than 7 to 8 d old calves. Furthermore, calves that were 13 to 24 d old at transport had lower haptoglobin (-0.16 g/L, 95% CI = -0.27 to -0.06) and potassium (-0.14 mmol/L, 95% CI = -0.23 to -0.05), and higher sodium (1.02 mmol/L, 95% CI = 0.22 to 1.82) and ionized calcium (0.02 mmol/L, 95% CI = 0.002 to 0.035) compared with calves transported at 7 to 8 d old. The results of this trial demonstrate that a rest period improves energy status upon arrival to a calf-raising facility, suggesting that the benefit of a rest period may be mostly related to the provision of mid-journey milk meals.