This study sought to compare early physiological and performance adaptations between a two-week cycle sprint interval training (SIT) and uphill run sprint training (UST) programs. Seventeen recreationally active adult males (age = 28 ± 5 years; body mass (BM) = 78 ± 9 kg) were assigned to either a control (n = 5), SIT (n = 6), or UST (n = 6) group. A discrete group of participants (n = 6, age = 33 ± 6 years, and body mass = 80 ± 9 kg) completed both training protocols to determine acute physiological responses. Intervention groups completed either a run or cycle peak oxygen uptake (VO2peak) test (intervention type dependent) prior to and following two weeks of training. Training comprised of three sessions per week of 4 × 30-s “all-out” sprints with a four-minute active recovery between bouts on a cycle ergometer against 7.5% of body mass in the SIT group and on a 10% slope in the UST group. The VO2peak values remained unchanged in both training groups, but time-to-exhaustion (TTE) was significantly increased only in the UST group (pre—495 ± 40 s, post—551 ± 15 s; p = 0.014) and not in the SIT group (pre—613 ± 130 s, post—634 ± 118 s, p = 0.07). Ventilatory threshold (VT) was significantly increased in both training groups (SIT group: pre—1.94 ± 0.45 L·min−1, post—2.23 ± 0.42 L·min−1; p < 0.005, UST group: pre—2.04 ± 0.40 L·min−1, post—2.33 ± 0.34 L·min−1, p < 0.005). These results indicate that UST may be an effective alternative to SIT in healthy individuals.