Rats

Eighteen male Wistar rats (Rattus norvegicus) (N = 18) were used in the study (4 months old). Rats were randomly divided into a control group and a nicotinamide riboside group (9 rats each). The animals were housed under a 12 h light:12 h dark cycle, controlled temperature (21–23 °C) and controlled humidity (50–70 %). Commercial rat chow (#160466; Biozois SA, Greece) and tap water were provided ad libitum. All rats were acclimatized in the animal facility and familiarized to swimming. All procedures were in accordance with the European Union guidelines for the care and use of laboratory animals, as well as the “Principles of laboratory animal care” (NIH publication No. 86–23, revised 1985). The project was reviewed and approved by the institutional review board and the appropriate state authority.

Nicotinamide riboside administration

Nicotinamide riboside (NAD+ Cell Regenerator™, Life Extension®, Fort Lauderdale, US) was administered daily for 21 days via gavage at a dose of 300 mg/kg body weight. The dose was chosen based on the study by Conze et al. [6], who reported that 300 mg/kg body weight of nicotinamide riboside did not cause any adverse events. The rats in the control group received a saline vehicle. Individual doses were freshly prepared daily for each rat separately just before the gavage procedure.

Incremental swimming performance test

The rats were removed from the vivarium to the swimming tanks immediately prior to the performance test after an overnight fast. Rats swam individually in water tanks. The performance test took place between 09:00 and 11:00. Rats swam individually until exhaustion at a water temperature of 34 °C. An incremental load was adjusted at the base of their tail. In particular, a load equal to 2 % of the rats’ body weight was adjusted for the first 4 min and then loads equal to 3.5 % and 5 % of the rats’ body weight were adjusted for the next 8 min (4 min each). A final load equal to 10 % of the rats’ body weight was used and rats were left to swim until exhaustion. A rat was considered to have reached exhaustion when it exhibited loss of coordinated movements and failure to return to the surface within 10 s three consecutive times.

Statistical analysis

A t-test for independent samples was performed to compare the time-to-exhaustion performance between the two groups (mean ± SD). To determine the meaningfulness of the difference in exercise performance between the groups, the effect size was calculated as the difference in time trial between the two groups divided by the mean standard deviation of the two groups. According to a modified Cohen scale (http://www.sportsci.org), effect sizes of 0.2, 0.6, 1.2, 2.0, and 4.0 were considered small, moderate, large, very large, and nearly perfect, respectively. For comparison, the values of the original Cohen scale are 0.2 for small, 0.5 for moderate, and 0.8 for large effects.