The aim of the present investigation was to investigate whether 6 weeks' high intensity interval training (HIIT; 6 x 30 s sprints at 40% peak power, once every five days) preceded by 6 weeks' aerobic preconditioning would affect fasting insulin, glucose, and the homeostatic model assessment of insulin resistance (HOMA1-IR) in older men. A secondary aim was to establish whether lifelong exercisers (LEX) exhibited improved fasting insulin, glucose, and HOMA1-IR, compared to sedentary older males (SED). Twenty-two males (62 ± 2 years) comprised the SED group and 17 males (60 ± 5 years) were enrolled as LEX. Participants were tested at phase A (baseline), B (after preconditioning), and C (post-HIIT). There was no effect of time (P=0.116) or interaction (P=0.727) on insulin. However, there was an effect of group (P<0.001). In terms of magnitude, HIIT induced a small decrease in SED insulin compared to baseline (15.8 ± 8.1 uIU·ml-1 at baseline and 14.0 ± 7.8 uIU·ml-1 post-HIIT; Cohen’s d=0.23) and compared to post-preconditioning (17.5 ± 9.7 uIU·ml-1; Cohen’s d=0.40). LEX insulin was unchanged throughout (all differences were trivial). Insulin was lower in LEX than SED at phase A (P<0.001, Cohen’s d=1.31), B (P=0.023, Cohen’s d=0.78), and C (P=0.004, Cohen’s d=1.01). There was no effect of time (P=0.290), group (P=0.166), or interaction (P=0.153) for glucose. In terms of magnitude, HIIT produced a small reduction in SED glucose compared to baseline (5.7 ± 1.3 mmol·l-1 at baseline and 5.3 ± 0.9 mmol·l-1 post-HIIT; Cohen’s d=0.36), and compared to phase B (5.6 ± 0.8 mmol·l-1; Cohen’s d=0.35). LEX glucose was unchanged throughout (all changes were trivial). SED had moderately higher blood glucose than LEX at phase A (Cohen’s d=0.49), and B (Cohen’s d=0.63), but only a trivial difference existed at phase C (Cohen’s d=0.15). There was no effect of time (P=0.110), or interaction (P=0.569) on HOMA1-IR. However, there was an effect of group (P=0.002). In terms of magnitude, SED HOMA1-IR was unchanged from phase A to B (4.2 ± 3.0 and 4.5 ± 2.9 arbitrary units respectively [Cohen’s d=0.10]). However, at C (3.5 ± 2.6) there was a small decrease compared to B (Cohen’s d=0.36), and A (Cohen’s d=0.25). LEX experienced a small increase in HOMA1-IR from phase A to B (1.6 ± 1.3 and 2.3 ± 2.8 respectively [Cohen’s d=0.32]), followed by a small decrease from B to C (1.7 ± 1.1 at phase C [Cohen’s d=0.28]), and a trivial change from A to C (Cohen’s d=0.08). HOMA1-IR was lower in LEX than SED at baseline (P=0.002, Cohen’s d=1.12), after preconditioning (P=0.024, Cohen’s d=0.77), and post-HIIT (P=0.014, Cohen’s d=0.90). Results of this study provide preliminary evidence that HIIT preceded by preconditioning results in small improvements in fasting insulin, glucose, and HOMA1-IR in SED. Yet, a larger confirmatory study is required to determine whether these effects translate into a larger sample.